sign in sign up
<<
Home , Invasive candidiasis, Mucorales, Mycosis, Pathogenesis

Current Fungal Reports (2019) 13:11–20 https://doi.org/10.1007/s12281-019-0337-1

CLINICAL (S CHALLA, SECTION EDITOR)

Mucormycosis: Pathogenesis and Pathology

Sundaram Challa1

Published online: 19 January 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract Purpose of Review is an opportunistic fungal infection associated with high mortality. Understanding the path- ogenesis and the resultant pathology in various organs enables to improve early diagnosis and treatment options. Recent Findings An immunocompetent host with intact skin/mucosal barrier and innate immunity is usually resistant to the infection; however, natural disasters and trauma account for the in healthy hosts. Neutropenia, , hyperglycemia, ketoacidosis, and other factors impair host defenses and make increased serum iron available to the for its growth. The has special iron assimilation mechanisms. The composition and genetic alterations allow rapid growth in host environment and evade host defenses. Expression of CotH proteins on the and hyphae facilitates adhesion to the receptors on endothelial cells, angioinvasion, and dissemination. Elaboration of lytic enzymes and proteases along with augment fungal invasion. Rhinocerebral mucormycosis is the most common clinical form. The pathology hall mark in various organs is angioinvasion resulting in hemorrhage, infarction, and suppurative . Summary The host defenses and how the risk factors alter and impair the host’s ability to prevent the invasion of the fungus are reviewed. The factors of the fungus to rapidly grow and disseminate in the host by evading recognition, suppressing immune response, manipulate the environment to derive nutrition, and disease are discussed.

Keywords Mucormycosis . Pathogenesis . Risk factors . mellitus . Host defenses . Virulence factors

Introduction

Mucormycosis is an emerging fungal infection associated Precise information on the prevalence of mucormycosis is with high mortality. The incidence is increasing due to lacking due to difficulties in clinical diagnosis. However, ac- increase in the number of organ transplantations, patients cording to the current worldwide reports, it is the third with , patients with diabetes mellitus (DM), and most common cause of invasive fungal following frequency of natural disasters and trauma [1••]. The prev- and with an increase of alence of severe cases of mucormycosis is high and the mucormycosis in the adult population and in infants less than poor prognosis of these infections is attributed to delay in 1yearold[3]. According to documented literature, the diagnosis and the inherent resistance of to mucormycosis is at least 5–10-fold less common than other most available [2]. Understanding the patho- fungal infections like aspergillosis [4, 5]. Differences in the genesis helps in developing better methods for early diag- epidemiology of mucormycosis seem to exist between devel- nosis and treatment strategies. oped and developing countries. While hematologic malignan- cies and hematopoietic stem cell transplantation (HSCT) are the leading causes of mucormycosis in the developed coun- tries, uncontrolled DM or trauma are the major causes in the This article is part of the Topical Collection on developing countries, especially in India [6–9, 10••].

* Sundaram Challa [email protected] Causative Agents 1 Department of Pathology, Basavatarakam Indo American Hospital and Research Institute, Hyderabad, Telangana State 500034, These organisms belong to the Mucorales of the sub- India phylum of the phylum after 12 Curr Fungal Infect Rep (2019) 13:11–20 the reclassification based on molecular characteristics [11]. v. injection drug use , as was originally described, included two clin- vi. prolonged hospital stay icopathologically different , mucormycosis and is entomophthoromycosis. However, molecular phylogenetic analysis confirmed the phylum to be polyphylet- Mode of Spread ic and hence does not include Zygomycota. The earlier term Zygomycosis hence has become obsolete [11]. The term The spores enter the body by mucormycosis refers to diseases caused by members of Mucorales. spp. are the most common causative I. inhalation agents, followed by , , , II. ingestion of contaminated food ,andCunninghamella [12•, 13]. They are ubiqui- III. implantation in injured skin by trauma//surgery, or tous with worldwide distribution and thermo-tolerant, grow- IV. percutaneous route by contaminated needles or catheters ing on decaying organic matter. The morphology shows [14, 15](Fig.1). aseptate/pauci septate broad (3–25 μm), ribbon-like, hyaline hyphae with irregular or right angle branching. Pathogenesis

Risk Factors The host-pathogen interaction is crucial in developing disease. To cause disease, fungal spores need to enter, evade the host Mucormycosis is usually an opportunistic infection with and germinate, leading to angioinvasion and specific risk factors, though a small percentage of infec- dissemination [17••]. tions occur in immunocompetent hosts [6, 14]. The individ- uals at highest risk of developing invasive disease are those who have decreased numbers (quantitative defects) of Host Defenses mononuclear and polymorphonuclear phagocytes as in neu- tropenia or who have diseases that impair the function A healthy host with intact skin/mucosal barrier and innate im- (qualitative defects) of phagocytes as in hyperglycemia/ munity is usually resistant to develop disease even though the acidosis or administration of [1••, 6, 10••, spores are present ubiquitously. The mucus layer of sinus/ 15, 16, 17••]. These risk factors impair host defenses and has anti-microbial properties and poor in nutrients. Moreover, permit growth and dissemination of fungus resulting in in- the host keeps the essential nutrient iron firmly bound to the vasive disease. serum proteins to restrict access to in a process The conditions include the following: called nutrient immunity and keeps an iron poor environment as a defense strategy against fungal invasion [18]. i. hematologic Mononuclear cells provide the host defense. Tissue macro- ii. hematopoietic stem cell transplantation (HSTC) phages phagocytose the inhaled spores and kill them. If any iii. solid organ malignancies spores escape the macrophages and germinate, the hyphae iv. solid induce chemotaxis of neutrophils, which damage the hyphae v. on high dose /immuno-suppression and phagocytose them. Neutrophils produce reactive oxygen vi. rheumatologic diseases metabolites, perforin, cationic peptides, and enzymes and kill vii. uncontrolled diabetes mellitus with ketoacidosis the spores and hyphae by oxidative cytotoxic effect [19–22, viii. metabolic acidosis 23•]. Neutrophils also produce pro-inflammatory ix. on deferoxamine therapy like tumor factor-alpha (TNF-α), interferon-gamma x. multiple transfusions (INF-γ), interleukin-1b (IL-1b), which in turn activate and xi. malnutrition, neonatal prematurity recruit other inflammatory cells. The phagocytes express pat- xii. prophylaxis with tern recognition receptors like toll-like receptors (TLR) which identify and bind to the pathogen-associated molecular pat- The infections that occur in immunocompetent individuals terns (PAMP) on the fungi [22]. This recognition activates are the following intracellular signaling and mediates inflammation [20]. Platelets play an important role in host immunity due to their i. skin injuries, burns, trauma antimicrobial and anti-fungal properties. They secrete granules that ii. natural calamities like tornadoes, tsunami, volcanic eruption have pro-inflammatory and anti-inflammatory cytokines and iii. combat-related injuries chemokines with fungicidal properties [23•]. They express mem- iv. contaminated bandages, tongue depressors brane bound molecules that bind to endothelial cells, monocytes, Curr Fungal Infect Rep (2019) 13:11–20 13

Fig. 1 Mucormycosis: modes of spread and clinical forms with common pathologic process

and dendritic cells leading to their activation [24, 25]. Platelets get maintaining growth and metabolism under highly varied envi- activated by adhering to Mucorales spores and hyphae leading to ronmental conditions, by the production of virulence factors, the aggregation and clot formation, resulting in suppression of capacity for accelerated fungal cell wall synthesis, and iron fungal growth and hematogenous dissemination [23•, 24]. assimilation [27, 28]. These genetic alterations allow the organ- Natural killer (NK) cells exert direct and indirect cytotoxic ism for rapid angio-invasive growth, evade and survive against effects and damage the hyphae of Mucorales.Theyproduce host immunity and hostile environment, derive nutrition from chemokines and cytokines like TNF-α,IFN-γ,and the host, and develop resistance to agents [29]. granulocyte-macrophage colony-stimulating factor (GM- The bigger size of the Mucorales gets them trapped CSF) and exert their effect on other immune cells [26]. in the paranasal sinus usually; however, the size is variable from 3 to 11 μm, depending on the and hence the spores can reach the lung also to cause disease [30••]. Altered Host Factors Mucorales are thermo-tolerant but no clear correlation was found between growth at host temperature and virulence [30••]. Stress tolerance and nutrient acquisition and utilization Neutropenia and immunosuppression hamper the host de- differ among species but are not related to virulence [31, 32]. fenses and allow growth of the fungus. Hyperglycemia with Other potential virulence factors include secretion of lytic ketoacidosis increases the risk for mucormycosis. Changes in enzymes as well as metabolites like alkaloids or mycotoxins, iron availability to the fungus may play an important role in which facilitate tissue invasion and inhibit host immune re- the pathogenesis of mucormycosis in diabetics with sponse. Mucorales have a number of genes that encode for the ketoacidosis. Administration of corticosteroids impairs host’s lytic enzymes. However, their direct involvement in human ability to prevent of spores, whereas exposure to cases of mucormycosis is yet to be documented [33]. voriconazole makes the organism highly virulent, possibly due to an epigenetic modification [19, 27]. Interaction with Epithelium

Virulence Factors of the Pathogen Much is not known about how Mucorales interact with epithe- lial cells. When there is damage to the epithelium, the spores Much of the research is focused on as that is adhere to the extracellular matrix proteins in the basement mem- the most common etiologic agent of mucormycosis. However, brane, laminin, and collagen IV by specific binding. Mucorales the virulence factors vary among species. The genome se- secrete a number of lipolytic/glycosidic enzymes, proteases quencing of Rhizopus oryzae has shown that in its evolution, (aspartic proteases), and subtilases, which contribute to the de- the organism has equipped itself with the capabilities for struction of the stroma and facilitate host invasion [34, 35]. 14 Curr Fungal Infect Rep (2019) 13:11–20

Interaction with Endothelium Hyperglycemia and Ketoacidosis Hyperglycemia with ketoacidosis in uncontrolled DM increases the risk of The Mucorales adhere to the endothelial cells by expressing mucormycosis. Hyperglycemia causes glycosylation of iron se- spore coat homolog (CotH) proteins [35]. The CotH proteins questering proteins transferrin, ferritin, and lactoferrin, reducing are exclusive to Mucorales and the virulence depends on the their iron affinity and cause proton mediated displacement of number of copies expressed (3–7 copies expressed in Rhizopus, ferric iron resulting in increased free iron levels even without Mucor,andLichtheimia which cause disease more frequently acidosis [33]. Hyperglycemia also causes phagocyte dysfunc- compared to 1–2copiesinApophysomyces, , tion with poorly characterized defects [14, 21].Thepresenceof ,andSyncephalastrum which cause disease less fre- ketone bodies like β-hydroxy butyrate [BHB] and the low pH quently) [36•]. The CotH proteins on the germlings bind to the in the blood vessels strongly impairs the ability of transferrin to receptor glucose-regulator protein 78 (GRP78) on the endothe- chelate iron [43]. The increased available serum iron is lial cells. GRP78 is a heat shock protein which is a unique host transported intracellularly by the reductase-permease system cell receptor [37]. The invasion is a two-step process that in- [1••, 44•]. Glucose, iron, and BHB induce the expression of volves adhesion and GRP78 triggered invasion [38]. The inter- GRP78 and CotH, and this enhanced expression results in the action and binding of CotH and GRP78 facilitate endocytosis growth of the fungus and augmented fungal invasion with sub- of the fungus into the endothelial cell resulting in its damage. sequent injury of the endothelium [38, 45]. These host factors This expression of CotH and GRP78 is enhanced by hypergly- suppress the T lymphocyte induction, IFN-γ production, and cemia and availability of iron in acidic pH. Endothelial damage phagocyte-mediated killing [45]. Ketoacidosis also impairs is also mediated by secondary metabolites produced by the chemotaxis and phagocytosis of neutrophils and facilitates dis- Mucorales which act as toxins. semination of the fungus [21, 30••, 33]. Fungal cell adherence and endocytosis both contribute to the ability of Mucorales strains to cause host . Acquisition of Iron by Using Siderophores R. orzyae lacks Platelet-derived growth factor receptor (PDGFR) has been genes for non-ribosomal peptide synthases, which are the en- shown to promote endocytosis [36•]. By in vitro studies, it zymes that produce the most common siderophores was shown that PDGFRB pathway was activated in infections (hydroxamate siderophores). The intrinsic siderophore, with Mucorales similar to infections with albicans to rhizoferrin secreted by Rhizopus, is inefficient in obtaining iron damage barrier host cells. Different fungi have common an- from the serum [46]. Hence, Mucorales are dependent on free cestor in the evolution and hence share common predicted iron in the serum as in diabetic ketoacidosis or the action of iron host signaling pathways [36•, 39]. PDGF regulates cell growth reductases or hemeoxygenases for iron uptake [29]. and division, particularly angioinvasion. Rhizopus likely in- Deferoxamine,anironchelatorusedinpersonswithincreased duces expression of angiogenesis pathways to aid in its hema- risk of iron overload, is a xenosiderophore and it directly che- togenous dissemination as it was shown that inhibitors to lates iron from transferrin, resulting in ferroxamine (iron-defer- PDGFRB (dimeric protein with two B subunits) reduce endo- oxamine complex). It binds to the surface of the fungus by thelial damage and angioinvasion [1••, 23•, 36•, 38, 40]. receptors, called Fob 1 and Fob 2 b and the fungus liberates ferrous iron from ferroxamine by reduction at the cell surface [44•]. The ferrous iron on the surface of the fungus is re-oxidized Growth of the Fungus to ferric iron by copper oxidase and then transported intracellu- larly by a high affinity iron permease, called FTR1 [46, 47]. For growth and replication, Mucorales derive nutrition from the host, manipulating its environment and disseminate by Hemoglobin as a Source of Iron Mucorales use hemoglobin as escaping recognition and immune attack by the host. a source of iron, and it has access to heme due to its angio- invasive nature. The fungus transports heme intracellularly Role of Iron Iron is essential for the growth of the fungus. and ferric iron is obtained by the action of heme-oxygenase However, in a normal host, iron is tightly bound to the serum in the cytoplasm. Alternatively, ferric iron from heme is ob- proteins. In animal models, elevated iron concentrations were tained by the action of reductase-permease system [46, 47]. shown to enhance the growth by inhibiting the function of phagocytes and decreasing IFN-γ secretion, whereas iron Evasion of Host Defenses Mucorales down regulate the genes deprivation resulted in apoptosis of the fungus [41, 42]. involved in pathogen recognition, innate immune defense Strategies for iron acquisition can be generally divided into mechanisms, and tissue repair mechanisms, thereby facilitat- siderophore, heme, and free iron acquisition systems. ing fungal growth [31, 32]. Siderophores are low molecular weight iron chelators that are secreted by bacteria/fungi and bind iron with an affinity Resistance to Antifungal Agents Resistance to antifungal that surpasses that of transferrin and lactoferrin [18]. agents is one of the important virulence factors of Curr Fungal Infect Rep (2019) 13:11–20 15

Mucorales. Recent studies showed that the pathogen is genet- rhinoorbital, pulmonary, cutaneous, gastrointestinal, dissemi- ically equipped for adaptation to hostile environments such as nated, or uncommon forms. the effects of antifungal agents or host-pathogen interaction. In a histopathologically verified series of 410 fungal infec- The antifungal resistance is mediated by Mendelian mutations tions during the period 1988 to 2012 in a university hospital in or epigenetic pathways [48]. South India, mucormycosis was the second commonest infec- Calcineurin is a fungal virulence factor which has a tion (following aspergillosis) constituting 107 (26.10%) cases central role in various fungal physiologic processes, in- (unpublished data). Risk factors were identified in 100 cluding pathways that counteract the damaging effects of (93.46%) patients with DM being the commonest (77%). antifungals and promote resistance. It governs the dimor- Rhino-cerebral form was the commonest mode of spread phic transition of the fungus [49]. Under experimental followed by direct implantation due to surgery/trauma. The conditions, when exposed to stressful compounds like involvement was disseminated in 2 (1.87%), rhino FK506, the drug forms a complex with FK506-binding orbitocerebral in 66 (61.68%), pulmonary in 8 (7.48%), cuta- protein, a peptidyl-prolyl isomerase FKBP12 present in neous in 20 (18.69%), gastro-intestinal in 7 (6.54%), focal, the pathogenic fungi and this complex inhibits calcine- isolated in kidney and bone 2 (1.87%) each. Diagnosis was urin. Mutations in the fkbA gene encoding FKBP12 confer established at autopsy only in 6 (5.61%) patients. FK506 resistance. Inhibition or mutation of calcineurin (cnbR or cnaA genes) or low oxygen/high carbon- Pathology Regardless of the route of entry or anatomical site dioxide in the milieu forces the fungus to be form, involved, the pathology hallmark of mucormycosis is hemor- whichislessvirulent[48–50, 51•]. rhage, thrombosis, infarction, and tissue necrosis. The inflam- RNA interference (RNAi) is a complex gene regulatory mation is predominantly neutrophilic, suppurating granulo- system that blocks the expression of antifungal drug target matous, or sparse/minimal [55]. The fungus is angioinvasive, genes and the fungi utilize these pathways in various cel- disseminates hematogenously causing multi-organ involve- lular processes to adapt to different environmental condi- ment [17••]. In addition to angioinvasion, the fungus also tions to rapidly generate drug-resistant epimutant strains spreads by retrograde extension along nerves and perineural [51•]. The different RNAi pathways suppress gene expres- invasion occurs concurrently with angioinvasion [56, 57] sion through a mechanism mediated by small non-coding (Fig. 2a–j). RNAs (sRNAs). The double-stranded RNA precursors produce sRNAs by the action of RNase III Dicer en- Rhinocerebral Mucormycosis This is the most common form zymes, which enter a RNA-induced silencing complex to of the disease and diabetic ketoacidosis/uncontrolled diabetes recognize the target RNA. Once the target RNA is cap- is the most common risk factor [6, 7, 10••, 58–62]. The infec- tured, it degrades the RNA or blocks its translation [51•, tion spreads from the mucosa of nose/nasopharynx and 52, 53]. Recently, a novel, non-canonical RNA-dependent spreads to adjacent soft tissues. Upon germination, the invad- RNA polymerase (RdRP)-dependent Dicer independent ing fungus may spread inferiorly to invade the palate, poste- degradation mechanism for endogenous mRNA was de- riorly to invade the sphenoid sinus, laterally into the cavernous scribedwhereDicerisreplacedbyanewRNAIIIlike sinus to involve the orbits, or cranially through orbital apex or protein, R3B2 [54]. This pathway exerts an inhibitory cribriform plate to invade the brain [63]. Due to the large size effect on the RNAi-mediated epimutation mechanism of the hyphae, they occlude larger vessels resulting in infarcts and controls the expression of genes via specific degrada- (Fig. 2a, b). The fungus is highly angiotropic and invades the tion of the corresponding target mRNAs. Hence, the target arteries in the orbit, internal carotid artery, and cavernous sinus RNA degradation may be mediated by canonical or non- and produces thrombosis and hemorrhage. The Mucorales canonical pathways. The interaction between the two produce acute fulminant fungal rhinosinusitis and hemorrhag- pathways is complex and competitive for the target ic necrosis at multiple sites in the brain, particularly at the base mRNA. Mutations in the genes (r3b2 and rdrp3)thatspe- of frontal lobes and deep gray nuclei [10••, 58–62]. cifically block the mRNA degradation pathway enhance Microscopically extensive areas of infarction with or with- the production of drug-resistant epimutants [51•]. out hemorrhage are seen with sparse neutrophilic infiltrate. These studies further our understanding of the mechanisms Multinucleated giant cells may be seen but well-formed deployed by fungal pathogens to survive and adapt under stress- granulomas are uncommon. The broad aseptate hyphae ful environmental conditions. Manipulating the RNAi pathways are seen invading vessels. Mycotic aneurysms are uncom- holds promise for developing new therapeutic targets [51•] monly reported [58, 60, 64]. Fungal hyphae may be seen in (Table 1). the perineurium, particularly, trigeminal nerve due to peri- neural spread of the fungus [56]. Perineural invasion is Clinical Forms of the Disease Depending on the anatomical reported along with angioinvasion and suggests extensive site of infection, mucormycosis is classified as rhinocerebral/ disease [55, 57](Fig.2a–d). 16 Curr Fungal Infect Rep (2019) 13:11–20

Table 1 Mucormycosis: normal host defenses, altered host factors and virulence factors of the pathogen

Normal host defenses Altered host factors Virulence of pathogen

Intact skin/mucosa Disrupted skin/mucosa Spores adhere to ECM proteins laminin, collagen Cilia trap the spores and mucus has antimicrobial IV; fungi are thermo-tolerant properties Temperature 37 °C Phagocytes: Macrophages phagocytose spores, DM makes macrophages dysfunctional and impairs Down regulate genes involved in pathogen prevent germination phagocytosis recognition, innate immune defense and tissue Neutrophils produce chemokines, cytokines, growth Neutropenia impairs host defenses and allows repair mechanisms. factors to damage and kill hyphae. growth of fungus Cell wall composition, genes for rapid synthesis of Identify PAMP on the fungi by pattern recognition Hyperglycemia suppresses the T lymphocyte cell wall allow evasion of phagocytosis; receptors like TLR-2 and mediate inflammation. induction, IFN- production, and expression of CotH facilitates endothelial phagocyte-mediated killing and ketoacidosis adhesion, damage. impairs chemotaxis and phagocytosis Genetic alterations allow fungus to evade host defenses, facilitate rapid growth, angio-invasion and dissemination. Produce lytic enzymes, secondary metabolites and mycotoxins which facilitate fungal invasion and suppress host immune response. Activates PDGF mediated signaling pathway to promote endocytosis, host cell damage, angioinvasion and dissemination. RNAi pathway blocks the expression of antifungal drug target genes and produces mutant strains by epigenetic mechanisms that resist and survive hostile environment factors and antifungal agents Platelets secrete chemokines and cytokines which are Thrombocytopenia facilitates fungal growth Suppresses host immune response fungicidal; express molecules to adhere to endothelial cells, monocytes and dendritic cells and activate them Adhesion and aggregation suppress angioinvasion and dissemination. NK cells adhere and damage fungal hyphae; produce Immunosuppression facilitates fungal growth Spores are not affected by NK cells; Inhibit IFN-γ chemokines and cytokines like TNF-α, IFN-γ and production and evade host defense GM-CSF which are cytotoxic Nutrients: Hyperglycemia induces glycosylation of ferritin and Fungus expresses CotH to adhere to the GRP78 Iron is tightly bound to serum proteins; normal transferrin and facilitates release of iron; ketone receptor on endothelial cells in DKA concentration of glucose; bodies lower pH; increased serum iron available in pH maintained at 7.4 DKA and during deferoxamine therapy; glucose, Fungus uses siderophores to acquire iron from iron, BHB induce expression of GRP78 and CotH ferroxamine and hemoglobin and transport which facilitate endothelial adhesion and intracellularly by expressing receptors and using endocytosis of fungus oxidase reductase system.

ECM extracellular matrix, DM diabetes mellitus, PA MP pathogen-associated molecular pattern, TLR-2 toll-like receptor-2, CotH proteins spore coat homolog proteins, IFN-γ interferon-gamma, NK cells natural killer cells, TNF-α tumor necrosis factor-alpha, GM-CSF granulocyte macrophage-colony stimulating factor, DKA diabetic ketoacidosis, BHB beta hydroxyl butyrate, GRP78 glucose-regulated protein 78, PDGF platelet-derived growth factor, RNAi ribonucleic acid interference

Pulmonary Mucormycosis Neutropenia, especially in patients [14, 33]. Patients have necrotic eschar, , or fasciitis [69, with hematologic malignancy or HSCT, is the most common 70]. It is locally invasive with involvement of subcutaneous risk factor followed by diabetes mellitus [6, 10••, 65, 66]. The tissue, adipose tissue, muscle, and fascia [71, 72]. pulmonary lesions may be infiltrates/mass lesions/cavitary Involvement of vessels may lead to hematogenous dissemina- lesions/ single or multiple nodules/consolidation or tracheitis tion to other organs and skin may be involved secondarily in [66, 67]. Microscopically, vascular invasion and hemorrhagic disseminated disease. Necrosis with suppurative inflammation infarction/hemorrhages are common. The less common and and infarction may be seen [10••, 14](Fig.2i). less specific features of pulmonary mucormycosis include bronchial invasion, , lung abscesses, and granulo- Gasreointestinal Mucormycosis Itcanoccurinbothimmuno- matous pneumonitis [65, 68](Fig.2h). competent and immunosuppressed individuals. Neonatal prema- turity, malnutrition, and diabetes mellitus are the common risk Cutaneous Mucormycosis Majority of the patients are immu- factors [6, 33, 73–76]. Stomach is the most common site of in- nocompetent who have disruption of skin barrier due to injury volvement, followed by colon and ileum, though any part of the Curr Fungal Infect Rep (2019) 13:11–20 17

Fig. 2 a Photomicrograph of rhinocerebral mucormycosis with broad aseptate hyphae beneath the sinus mucosa. H&E × 10; inset showing vessel occluded by fungal hyphae. GMS × 10. b Right temporo parietal hemorrhagic infarct. c Angioinvasion with hyphae in the vessel wall and lumen. GMS × 40. d Perineural invasion with hyphae in the perineurium. GMS ×40.e Gross specimen of stomach with necrotic ulcer. f Gross specimen of jejunum with hemorrhagic discoloration. g Photomicrograph of small intestine with H&E × 10; inset shows hyphae in the vessel wall. GMS × 10 (H&E: hematoxylin and eosin; GMS: Gomori methenamine silver)

gastro intestinal tract may be involved. It may also involve liver, Acknowledgements The author acknowledges the faculty, residents and ’ spleen, and pancreas [77]. In premature neonates, it causes necro- staff of department of Pathology, Nizam s Institute of Medical Sciences, Hyderabad, India. tizing enterocolitis whereas in neutropenic patients, it forms mass lesions. Microscopically, it shows angioinvasion leading to hem- Compliance with Ethical Standards orrhage, gangrene, perforation, and peritonitis [78, 79](Fig.2e–g). Conflict of Interest The authors declare that they have no conflicts of Disseminated Mucormycosis It involves two or more non- interest. contiguous organs and lung is the most common organ in- volved. Patients with iron overload and severe immunosup- Human and Animal Rights and Informed Consent This article does not pression develop disseminated disease [10••]. The infection contain any studies with human or animal subjects performed by any of the authors. may spread from lung, GIT, or skin to other organs by hema- togenous dissemination. Microscopically, angioinvasion with Publisher’sNote Springer Nature remains neutral with regard to jurisdic- hemorrhagic infarcts and necrosis are seen [75, 80]. tional claims in published maps and institutional affiliations.

Uncommon Forms of Mucormycosis Intravenous drug use is the most common risk factor. The involvement may be oste- References omyelitis, pyelonephritis, , peritonitis, or focal in- volvement of any organ [10••, 80](Fig.2j). Papers of particular interest, published recently, have been highlighted as: • Of importance Conclusion •• Of major importance

Patients with neutropenia, diabetic ketoacidosis, and those on 1.•• Baldin C, Ibrahim AS. Molecular mechanisms of mucormycosis-The deferoxamine therapy are at increased risk of developing bitter and the sweet. PLoS pathog. 2017;13(8):e1006408. https://doi. mucormycosis. The virulence factors involved in the host org/10.1371/journal.ppat.1006408 Excellent review on the host pathogen interaction in the at risk host lead to angioinvasion pathogen interactions in mucormycosis and how the altered host factors like hyperglycemia and ketoacidosis lead to and pathology in various organs. Correction of the predispos- expression of receptors on endothelial cells and ligands on the ing conditions, augmenting host defenses and counter the vir- pathogen, resulting in adhesion, angioinvasion and ulence of the pathogen, offer promise for better outcome. dissemination of the fungus. 18 Curr Fungal Infect Rep (2019) 13:11–20

2. Tissot F, Agrawal S, Pagano L, Petrikkos G, Groll AH, Skiada A, which have implications in therapeutic interventions. The role et al. ECIL-6 guidelines for the treatment of , of phagocytes, platelets and NK cells in host defenses and the aspergillosis and mucormycosis in leukemia and hematopoietic mechanisms of iron uptake, pathogen-endothelial interaction, stem cell transplant patients. Haematologica. 2017;102(3):433–44. angioinvasion and dissemination are discussed in detail. https://doi.org/10.3324/haematol.2016.152900. 18. Hood MI, Skaar EP. Nutritional immunity: transition metals at the 3. Dignani MC. Epidemiology of invasive fungal diseases on the basis pathogen-host interface. Nat Rev Microbiol. 2012;10(8):525–37. of autopsy reports. F1000Prime Rep. 2014;6:81. Published 2014 https://doi.org/10.1038/nrmicro2836 Review. Sep 4. https://doi.org/10.12703/P6-81. 19. Lionakis MS, Kontoyiannis DP. Glucocorticoids and invasive fun- 4. Kontoyiannis DP, Lionakis MS, Lewis RE, Chamilos G, Healy M, gal infections. Lancet. 2003;362(9398):1828–38 Review. Perego C, et al. Zygomycosis in a tertiary-care cancer center in the era 20. Chamilos G, Lewis RE, Lamaris G, Walsh TJ, Kontoyiannis DP. of -active antifungal therapy: a case-control observational Zygomycetes hyphae trigger an early, robust proinflammatory re- study of 27 recent cases. J Infect Dis. 2005;191(8):1350–60. sponse in human polymorphonuclear neutrophils through toll-like 5. Chamilos G, Luna M, Lewis RE, Bodey GP, Chemaly R, Tarrand receptor 2 induction but display relative resistance to oxidative JJ, et al. Invasive fungal infections in patients with hematologic damage. Antimicrob Agents Chemother. 2008;52(2):722–4. malignancies in a tertiary care cancer center: an autopsy study over 21. Ibrahim AS, Spellberg B, Walsh TJ, Kontoyiannis DP. – a 15-year period (1989-2003). Haematologica. 2006;91(7):986 9. Pathogenesis of mucormycosis. Clin Infect Dis. 2012;54(Suppl 6. Roden MM, Zaoutis TE, Buchanan WL, Knudsen TA, Sarkisova 1):S16–22. https://doi.org/10.1093/cid/cir865 Review. TA, Schaufele RL, et al. Epidemiology and outcome of 22. Roilides E, Kontoyiannis DP, Walsh TJ. Host defenses against zygomycosis: a review of 929 reported cases. Clin Infect Dis. zygomycetes. Clin Infect Dis. 2012;54(Suppl 1):S61–6. https:// – 2005;41(5):634 53 Review. doi.org/10.1093/cid/cir869 Review. 7. Chakrabarti A, Das A, Mandal J, Shivaprakash MR, George VK, 23.• Ibrahim AS, Voelz K. The mucormycete-host interface. Curr Opin Tarai B, et al. The rising trend of invasive zygomycosis in patients – – Microbiol. 2017;40:40 5. https://doi.org/10.1016/j.mib.2017.10.010 with uncontrolled diabetes mellitus. Med Mycol. 2006;44(4):335 42. Review. This review discusses the host pathogen interactions with 8. Chakrabarti A, Singh R. The emerging epidemiology of mould infec- – particular emphasis on host defenses and risk fators. The iron tions in developing countries. Curr Opin Infect Dis. 2011;24(6):521 6. acquisition mechanisms of the pathogen are discussed. https://doi.org/10.1097/QCO.0b013e32834ab21e Review. 24. Perkhofer S, Kainzner B, Kehrel BE, Dierich MP, Nussbaumer W, 9. Skiada A, Pagano L, Groll A, Zimmerli S, Dupont B, Lagrou K, Lass-Flörl C. Potential antifungal effects of human platelets against et al. European Confederation of Medical Working zygomycetes in vitro. J Infect Dis. 2009;200(7):1176–9. https://doi. Group on Zygomycosis. Zygomycosis in Europe: analysis of 230 org/10.1086/605607. cases accrued by the registry of the European Confederation of 25. Speth C, Rambach G, Lass-Flörl C. Platelet immunology in fungal Medical Mycology (ECMM) working group on Zygomycosis be- infections. Thromb Haemost. 2014;112(4):632–9. https://doi.org/ tween 2005 and 2007. Clin Microbiol Infect. 2011;17(12):1859–67. 10.1160/TH14-01-0074 Review. https://doi.org/10.1111/j.1469-0691.2010.03456.x. 10.•• Petrikkos G, Skiada A, Lortholary O, Roilides E, Walsh TJ, 26. Schmidt S, Tramsen L, Perkhofer S, Lass-Flörl C, Hanisch M, Kontoyiannis DP. Epidemiology and clinical manifestations of Röger F, et al. Rhizopus oryzae hyphae are damaged by human – natural killer (NK) cells, but suppress NK cell mediated immunity. mucormycosis. Clin Infect Dis. 2012;54(Suppl 1):S23 34. https:// – doi.org/10.1093/cid/cir866 Review. Excellent review on the Immunobiology. 2013;218(7):939 44. https://doi.org/10.1016/j. epidemiology, risk factors, mode of spread and clinical imbio.2012.10.013. manifestations of mucormycosis. 27. Lamaris GA, Ben-Ami R, Lewis RE, Chamilos G, Samonis G, 11. Kwon-Chung KJ. of fungi causing mucormycosis and Kontoyiannis DP. Increased virulence of Zygomycetes organisms (zygomycosis) and nomenclature of the dis- following exposure to voriconazole: a study involving fly and mu- – ease: molecular mycologic perspectives. Clin Infect Dis. rine models of zygomycosis. J Infect Dis. 2009;199(9):1399 406. 2012;54(Suppl 1):S8–S15. https://doi.org/10.1093/cid/cir864 Review. https://doi.org/10.1086/597615. 12.• Park HR, Voigt K. Interaction of Zygomycetes with innate immune 28. Ma L-J, Ibrahim AS, Skory C, et al. Genomic analysis of the basal cells reconsidered with respect to ecology, morphology, evolution lineage fungus rhizopus oryzae reveals a whole-genome duplica- and infection biology: a mini-review. Mycoses. 2014;57(Suppl 3): tion. Madhani HD, ed. PLoS Genet. 2009;5:e1000549. 31–9. https://doi.org/10.1111/myc.12235 Review. Areviewonthe 29. Lewis RE, Lortholary O, Spellberg B, Roilides E, Kontoyiannis DP, molecular classification of Zygomycetes and the separation of Walsh TJ. How does antifungal pharmacology differ for Mucorales from other genera. The review focuses on the innate mucormycosis versus aspergillosis? Clin Infect Dis. immunity of the host against infection by Mucorales. 2012;54(Suppl 1):S67–72. https://doi.org/10.1093/cid/cir884. 13. Farmakiotis D, Kontoyiannis DP. Mucormycoses. Infect Dis 30.•• Binder U, Maurer E, Lass-Flörl C. Mucormycosis–from the patho- Clin N Am. 2016;30(1):143–63. https://doi.org/10.1016/j.idc. gens to the disease. Clin Microbiol Infect. 2014;20(Suppl 6):60–6. 2015.10.011 Review. https://doi.org/10.1111/1469-0691.12566 Review. Excellent 14. Spellberg B, Edwards J Jr, Ibrahim A. Novel perspectives on review on the virulence of Mucorales in causing disease. The mucormycosis:pathophysiology, presentation, and management. taxonomic classification, virulence factors, angioinvasion Clin Microbiol Rev. 2005;18(3):556–69 Review. mechanisms and iron acquisition from host are described in 15. Sun HY, Singh N. Mucormycosis: its contemporary face and man- detail. The different clinical forms of the disease and their agement strategies. Lancet Infect Dis. 2011;11(4):301– 11. https:// manifestations are discussed. doi.org/10.1016/S1473-3099(10)70316-9 Review. 31. Alastruey-Izquierdo A, Hoffmann K, de Hoog GS, Rodriguez- 16. Neblett Fanfair R, Benedict K, Bos J, Bennett SD, Lo YC, Adebanjo Tudela JL, Voigt K, Bibashi E, et al. Species recognition and clin- T, et al. Necrotizing cutaneous mucormycosis after a tornado in ical relevance of the zygomycetous Lichtheimia (syn. Joplin, Missouri, in 2011. N Engl J Med. 2012;367(23):2214–25. pro parte, Mycocladus). J Clin Microbiol. 2010;48(6): 17.•• Petrikkos G, Tsioutis C. Recent Advances in the Pathogenesis of 2154–70. https://doi.org/10.1128/JCM.01744-09. Mucormycoses. Clin Ther. 2018;(6):40, 894–902. https://doi.org/ 32. Morace G, Borghi E. Invasive infections: virulence and path- 10.1016/j.clinthera.2018.03.009 Review. Excellent review on the ogenesis of mucorales. Int J Microbiol. 2012;2012:349278. https:// recent developments in the pathogenesis of mucormycosis doi.org/10.1155/2012/349278. Curr Fungal Infect Rep (2019) 13:11–20 19

33. Ribes JA, Vanover-Sams CL, Baker DJ. Zygomycetes in human 49. Lee SC, Li A, Calo S, Inoue M, Tonthat NK, Bain JM, et al. disease. Clin Microbiol Rev. 2000;13(2):236–301 Review. Calcineurin orchestrates dimorphic transitions, antifungal drug re- 34. Schoen C, Reichard U, Monod M, Kratzin HD, Rüchel R. sponses and host-pathogen interactions of the pathogenic Molecular cloning of an extracellular aspartic proteinase from mucoralean fungus . Mol Microbiol. and evidence for its expression during in- 2015;97(5):844–65. https://doi.org/10.1111/mmi.13071. fection. Med Mycol. 2002;40(1):61–71. 50. Lee SC, Li A, Calo S, Heitman J. Calcineurin plays key roles in the 35. Spreer A, Rüchel R, Reichard U. Characterization of an extracellu- dimorphic transition and virulence of the human pathogenic lar subtilisin protease of Rhizopus microsporus and evidence for its zygomycete Mucor circinelloides. Andrianopoulos A, ed. PLoS expression during invasive rhinoorbital . Med Mycol. Pathog. 2013;9:e1003625. 2006;44(8):723–31. 51.•• Calo S, Nicolás FE, Lee SC, Vila A, Cervantes M, Torres-Martinez 36.• Chibucos MC, Soliman S, Gebremariam T, Lee H, Daugherty S, Orvis S, et al. A non-canonical RNA degradation pathway suppresses J, et al. An integrated genomic and transcriptomic survey of RNAi-dependent epimutations in the human fungal pathogen mucormycosis-causing fungi. Nat Commun. 2016;7:12218. https:// Mucor circinelloides. PLoS Genet. 2017;13(3):e1006686. https:// doi.org/10.1038/ncomms12218 A review based on experimental doi.org/10.1371/journal.pgen.1006686 An excellent paper on the studies on the genetic basis of virulence factors. The article epigenetic pathway involving RNAi for antifungal resistance. brings about correlation between the number of CotH protein on 52. Chang SS, Zhang Z, Liu Y. RNA interference pathways in fungi: the pathogen species and frequency of clinical invasive disease. mechanisms and functions. Annu Rev Microbiol. 2012;66:305–23. 37. Roilides E, Antachopoulos C, Simitsopoulou M. Pathogenesis and https://doi.org/10.1146/annurev-micro-092611-150138 Review. host defence against Mucorales: the role of cytokines and interac- 53. Nicolás FE, Ruiz-Vázquez RM. Functional diversity of RNAi- – tion with antifungal drugs. Mycoses. 2014;57(Suppl 3):40–7. associated sRNAs in fungi. Int J Mol Sci. 2013;14(8):15348 60. https://doi.org/10.1111/myc.12236 Review. https://doi.org/10.3390/ijms140815348.Review. 38. Liu M, Spellberg B, Phan QT, Fu Y, Fu Y, Lee AS, et al. The 54. Trieu TA, Calo S, Nicolás FE, Vila A, Moxon S, Dalmay T, et al. A endothelial cell receptor GRP78 is required for mucormycosis path- non-canonical RNA silencing pathway promotes mRNA degrada- ogenesis in diabetic mice. J Clin Invest. 2010;120(6):1914–24. tion in basal Fungi. PLoS Genet. 2015;11(4):e1005168. https://doi. https://doi.org/10.1172/JCI42164. org/10.1371/journal.pgen.1005168. 39. Liu Y,Shetty AC, Schwartz JA, Bradford LL, Xu W, Phan QT, et al. 55. Cornely OA, Arikan-Akdagli S, Dannaoui E, Groll AH, Lagrou K, New signaling pathways govern the host response to C. albicans Chakrabarti A, et al. European Society of Clinical infection in various niches. Genome Res. 2015;25(5):679–89. and Infectious Diseases Fungal Infection Study Group; European https://doi.org/10.1101/gr.187427.114. Confederation of Medical Mycology. ESCMID and ECMM joint clinical guidelines for the diagnosis and management of 40. Kovalenko M, Rönnstrand L, Heldin CH, Loubtchenkov M, Gazit A, mucormycosis 2013. Clin Microbiol Infect. 2014;20(Suppl 3):5– Levitzki A, et al. Phosphorylation site-specific inhibition of platelet- 26. https://doi.org/10.1111/1469-0691.12371. derived growth factor beta-receptor autophosphorylation by the receptor 56. Frater JL, Hall GS, Procop GW. Histologic features of blocking tyrphostin AG1296. Biochemistry. 1997;36(21):6260–9. zygomycosis: emphasis on perineural invasion and fungal mor- 41. Cantinieaux B, Janssens A, Boelaert JR, Lejeune M, Vermylen C, phology. Arch Pathol Lab Med. 2001;125(3):375–8. Kerrels V,et al. Ferritin-associated iron induces neutrophil dysfunc- 57. Sravani T, Uppin SG, Uppin MS, Sundaram C. Rhinocerebral tion in hemosiderosis. J Lab Clin Med. 1999;133(4):353–61. mucormycosis: pathology revisited with emphasis on perineural 42. Shirazi F, Kontoyiannis DP, Ibrahim AS. Iron starvation induces – – spread. Neurol India. 2014;62:383 6. https://doi.org/10.4103/ apoptosis in Rhizopus oryzae in vitro. Virulence. 2015;6(2):121 0028-3886.141252. 6. https://doi.org/10.1080/21505594.2015.1009732. 58. Sundaram C, Mahadevan A, Laxmi V, Yasha TC, Santosh V, 43. Artis WM, Fountain JA, Delcher HK, Jones HE. A mechanism of Murthy JM, et al. Cerebral zygomycosis. Mycoses. 2005;48(6): susceptibility to mucormycosis in diabetic ketoacidosis: transferrin 396–407. – and iron availability. Diabetes. 1982;31(12):1109 14. 59. Sundaram C, Umabala P, Laxmi V, Purohit AK, Prasad VS, • 44. Liu M, Lin L, Gebremariam T, Luo G, Skory CD, French SW, et al. Panigrahi M, et al. Pathology of fungal infections of the central Fob1 and Fob2 Proteins Are Virulence Determinants of Rhizopus nervous system: 17 years' experience from Southern India. oryzae via Facilitating Iron Uptake from Ferrioxamine. PLoS . 2006;49(4):396–405. Pathog. 2015;11(5):e1004842. https://doi.org/10.1371/journal. 60. Shankar SK, Mahadevan A, Sundaram C, Sarkar C, Chacko G, ppat.1004842 A very good review on how Rhizopus oryzae Lanjewar DN, et al. Pathobiology of fungal infections of the central expresses receptors Fob 1 and Fob 2 to derive iron from nervous system with special reference to the Indian scenario. feroxamine in patients treated with deferoxamine and Neurol India. 2007;55(3):198–215 Review. transports iron intracellularly by iron permease FTR1. 61. Das A, Bal A, Chakrabarti A, Panda N, Joshi K. Spectrum of fungal 45. Gebremariam T, Lin L, Liu M, Kontoyiannis DP, French S, rhinosinusitis; histopathologist's perspective. Histopathology. Edwards JE Jr, et al. Bicarbonate correction of ketoacidosis alters 2009;54(7):854–9. https://doi.org/10.1111/j.1365-2559.2009.03309.x. host-pathogen interactions and alleviates mucormycosis. J Clin 62. Challa S, Uppin SG, Hanumanthu S, Panigrahi MK, Purohit AK, Invest. 2016;126(6):2280–94. https://doi.org/10.1172/JCI82744. Sattaluri S, et al. Fungal rhinosinusitis: a clinicopathological study 46. de Locht M, Boelaert JR, Schneider YJ. Iron uptake from from South India. Eur Arch Otorhinolaryngol. 2010;267(8):1239– ferrioxamine and from ferrirhizoferrin by germinating spores of 45. https://doi.org/10.1007/s00405-010-1202-6. Rhizopus microsporus. Biochem Pharmacol. 1994;47(10):1843–50. 63. Hosseini SM, Borghei P. Rhinocerebral mucormycosis: pathways 47. Ibrahim AS, Gebremariam T, Lin L, Luo G, Husseiny MI, Skory CD, of spread. Eur Arch Otorhinolaryngol. 2005;262(11):932–8. et al. The high affinity iron permease is a key virulence factor required 64. Sehgal A, Raghavendran M, Kumar D, Srivastava A, Dubey D, for Rhizopus oryzae pathogenesis. Mol Microbiol. 2010;77(3):587– Kumar A. Rhinocerebral mucormycosis causing basilar artery an- 604. https://doi.org/10.1111/j.1365-2958.2010.07234.x. eurysm with concomitant fungal colonic perforation in renal allo- 48. Calo S, Shertz-Wall C, Lee SC, Bastidas RJ, Nicolás FE, Granek graft recipient: a case report. Transplantation. 2004;78(6):949–50. JA, et al. Antifungal drug resistance evoked via RNAi-dependent 65. Challa S, Uppin SG, Uppin MS, Paul RT, Prayaga AK, Rao MT. epimutations. Nature. 2014;513(7519):555–8. https://doi.org/10. Pulmonary zygomycosis: a clinicopathological study. Lung India. 1038/nature13575. 2011;28(1):25–9. https://doi.org/10.4103/0970-2113.76297. 20 Curr Fungal Infect Rep (2019) 13:11–20

66. Yamin HS, Alastal AY, Bakri I. Pulmonary Mucormycosis over 130 73. Kontoyiannis DP, Lewis RE. Invasive zygomycosis: update on years: a case report and literature review. Turk Thorac J. 2017;18(1): pathogenesis,clinical manifestations, and management. Infect Dis 1–5. https://doi.org/10.5152/TurkThoracJ.2017.16033 Review. Clin N Am. 2006;20(3):581–607 vi. Review. 67. Wang XM, Guo LC, Xue SL, Chen YB. Pulmonary mucormycosis: 74. Shiva Prasad BN, Shenoy A, Nataraj KS. Primary gastrointestinal a case report and review of the literature. Oncol Lett. 2016;11(5): mucormycosis in an immunocompetent person. J Postgrad Med. 3049–53. 2008;54(3):211–3. 68. Walsh TJ, Gamaletsou MN, McGinnis MR, Hayden RT, 75. Uppin MS, Anuradha S, Uppin SG, Paul TR, Prayaga AK, Kontoyiannis DP. Early clinical and laboratory diagnosis of inva- Sundaram C. Fungal infections as a contributing cause of death: sive pulmonary, extrapulmonary, and disseminated mucormycosis an autopsy study. Indian J Pathol Microbiol. 2011;54:344–9. – (zygomycosis). Clin Infect Dis. 2012;54(Suppl 1):S55 60. https:// 76. Siu KL, Lee WH. A rare cause of intestinal perforation in an ex- doi.org/10.1093/cid/cir868. treme low birth weight infant-gastrointestinal mucormycosis: a case 69. Lakshmi V, Rani TS, Sharma S, Mohan VS, Sundaram C, Rao RR, report. J Perinatol. 2004; 24(5);319–21. et al. Zygomycotic necrotizing fasciitis caused by Apophysomyces – 77. Spellberg B. Gastrointestinal mucormycosis: an evolving disease. elegans. J Clin Microbiol. 1993;31(5):1368 9. Gastroenterol Hepatol (N Y). 2012;8(2):140–2. 70. Prasad RM, Bose SM, Vaiphei K, Verma GR. Post operative ab- 78. VirkSS,SinghRP,AroraAS,GrewalJS,PuriH.Gastric dominal wall mucormycosis mimicking as bacterial necrotising fas- zygomycosis—an unusual cause of massive upper gastrointestinal ciitis. J Postgrad Med. 2003;49(2):187–8. bleed. Indian J Gastroenterol. 2004;23(4):146–7. 71. de Oliveira-Neto MP, Da Silva M, Fialho Monteiro PC, Lazera M, de Almeida PR, Novellino AB, et al. Cutaneous mucormycosis in a 79. Geramizadeh B, Modjalal M, Nabai S, Banani A, Forootan HR, – Hooshdaran F, et al. Gastrointestinal zygomycosis: a report of three young, immunocompetent girl. Med Mycol. 2006;44(6):567 70 – Erratum in: Med Mycol. 2006 ;44(8):793. cases. Mycopathologia. 2007;164(1):35 8. 72. Li H, Hwang SK, Zhou C, Du J, Zhang J. Gangrenous cutaneous 80. Challa S, Prayaga AK, Vemu L, Sadasivan J, Murthy Jagarlapudi mucormycosis caused by Rhizopus oryzae: a case report and review MK, Digumarti, et al. Fungal endocarditis: an autopsy study. – of primary cutaneous mucormycosis in China over past 20 years. Asian Cardiovasc Thorac Ann. 2004;12:95 8. https://doi.org/10. Mycopathologia. 2013;176(1–2):123–8. https://doi.org/10.1007/ 1177/021849230401200202. s11046-013-9654-z Review.