Histoplasma Capsulatum: Infecção Pulmonar, Imunidade e Mecanismos de Defesa

Histoplasma capsulatum lung infection and immunity Michael C Horwath12 Roger A Fecher12 and George S Deepe Jr13 1Division of Infectious Diseases University of Cincinnati College of Medicine 3230 Eden Ave Cincinnati OH 45267 USA 2Division of Immunobiology Cincinnati Childrens Hospital Medical Center University of Cincinnati 2600 Clifton Ave Cincinnati OH 45220 USA 3Medical Service Veterans Affairs Hospital Cincinnati OH 45220 USA Abstract Histoplasma capsulatum an environmental fungus is the most common endemic pulmonary mycosis in the USA Disease is most frequently observed in immunocompromised patients living in endemic areas We present the mechanisms of fungal recognition innate immune response and adaptive immune response that lead to protection or exacerbation of disease Current understanding of these mechanisms is the result of a continuing dialogue between clinical observations and murine studies Mice are a powerful model to study the immune response to H capsulatum alone or in the presence of immunomodulatory drugs Vigilance for histoplasmosis should be exercised with novel immunosuppressive agents that target the important immune pathways identified here Keywords chemokines cytokines dendritic cells fungal disease Histoplasma capsulatum macrophages T cellmediated immunity Histoplasma capsulatum a global fungal pathogen Infection with Histoplasma capsulatum causes significant morbidity and mortality worldwide This fungus is a dimorphic ascomycete that grows in its hyphal form in soil and bird and bat guano Upon inhalation of spores H capsulatum transforms into the pathogenic yeast phase This form replicates within macrophages that carry the yeast from lungs to virtually any organ 1 2 Induction of adaptive immunity particularly the Th1 response is required for activation of macrophages and efficient clearance of the yeast Exposure to H capsulatum usually results in symptomless clearance however histoplasmosis can manifest as an acute flulike pulmonary illness a chronic cavitary lung disease or a progressive Author for correspondence georgedeepeucedu Authors contributed equally Financial competing interests disclosure The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed No writing assistance was utilized in the production of this manuscript HHS Public Access Author manuscript Future Microbiol Author manuscript available in PMC 2016 April 01 Published in final edited form as Future Microbiol 2015 June 10 967975 doi102217fmb1525 Author Manuscript Author Manuscript Author Manuscript Author Manuscript disseminated form 1 Immunocompromised individuals are especially at risk for disseminated infection the HIV pandemic resulted in a dramatic increase in lethal histoplasmosis Today highly active antiretroviral therapy HAART reduces this risk 2 Clinical disease develops in immunocompetent individuals with outbreaks occurring when there is a localized exposure to a large infectious dose 3 Distribution variation Cases of histoplasmosis occur worldwide but are concentrated in endemic regions The North American area is centered on the Ohio and Mississippi river valleys Large surveys of skin testing in the 1960s revealed that 80 of young adult men from this region were positive for H capsulatum exposure 1 4 Disease burden is regional an analysis of US hospital records estimated 3370 inpatient stays and 254 deaths associated with histoplasmosis in 2002 with almost 90 of hospitalizations occurring in midwestern and southern states 5 Central and South America contain large endemic areas and reviews have brought attention to the underdiagnosed burden of HIVassociated histoplasmosis in these regions 6 Additional endemic foci are in China southeast Asia the Indian subcontinent Australia and Africa 7 H capsulatum isolates exhibit considerable genotypic and phenotypic variability between and within these regions Genetic analysis has revealed that H capsulatum is not monophyletic and can be classified into seven or eight distinct clades 8 Genetic differences between clades lead to differences in phenotype and virulence Most isolates of H capsulatum have both α and βglucan in their cell walls but isolates from the North American 2 clade lack αglucan 9 While αglucan strains account for the majority of infections in immunocompetent individuals in North America αglucan infections are associated with HIV 10 In mouse models αglucan yeast causes more severe disease than αglucan but only at high infectious inocula 11 Thus strains of H capsulatum may depend on factors such as infectious dose or defective adaptive immune response to establish infection H capsulatum virulence H capsulatum is not contagious Despite infection usually being a dead end for fungal replication H capsulatum appears specifically adapted to mammalian hosts The transformation from mycelial to yeast phase at 37C is crucial for infection strains lacking this ability are avirulent 10 The yeast is equipped for evading intracellular killing by phagocytes with mechanisms to degrade reactive oxygen species ROS regulate lysosomal pH and capture essential nutrients that might otherwise be deprived 1215 Many mammalian species are infected with H capsulatum and are accidental hosts that must cope with the yeasts capability for survival within macrophages In both human and mouse infection macrophages provide a niche for H capsulatum proliferation that cannot be halted without adaptive immunity Horwath et al Page 2 Future Microbiol Author manuscript available in PMC 2016 April 01 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Innate immune response Recognition phagocytosis Immune cells use a variety of surface receptors to recognize and ingest H capsulatum Pattern recognition receptors such as Ctype lectin receptors and toll like receptors are necessary for innate immune cytokine production andor phagocytosis of pathogenic fungi 16 17 Dectin1 recognition of β13glucan is required for optimal proinflammatory cytokine production but not phagocytosis of H capsulatum αglucan on H capsulatum masks βglucan recognition 18 19 In humans a rare mutation that decreases surface expression of dectin1 is not associated with a higher incidence of histoplasmosis This suggests that dectin1 is not needed to combat infection or that these patients have not come in contact with the fungus 20 Other Ctype lectin receptors such as dectin2 and mincle bind H capsulatum but their role in immunity has not been elucidated 21 While macrophages and dendritic cells DCs exhibit overlapping expression of many surface receptors those utilized for phagocytosis of H capsulatum are cell specific Macrophages bind and ingest yeasts via CD11CD18 integrins while DCs utilize VLA5 to recognize H capsulatum ligands heat shock protein 60 and cyclophilin A respectively 22 24 CD11CD18 blockade reduces but does not prevent H capsulatum uptake by both human and murine macrophages this finding suggests that other receptors are capable of driving phagocytosis at least in the absence of CD11CD18 In contrast to macrophages human DCs rely on VLA5 for fungal recognition 25 Differential recognition of H capsulatum by macrophages and DCs may trigger unique signaling cascades CD11bCD18 triggers activation of the tyrosine kinase Syk and downstream production of proinflammatory cytokines in macrophages 19 VLA5 on the other hand activates kinases that regulate proliferation and survival including ERKMAPK and PI3KAkt This leads to production of antiapoptotic Bcl2 which may prevent fungaldriven apoptosis 26 Thus engagement of different receptors on these phagocytes may account for the contrasting intracellular fate Antifungal activity in neutrophils macrophages Following H capsulatum recognition appropriate cell mobilization is required for an effective immune response Neutrophils and macrophages are recruited early to the site of infection Human neutrophils are fungistatic not fungicidal against H capsulatum 27 This activity relies on cathepsin G defensins and bactericidalpermeabilityincreasing protein within azurophil granules 27 Neutrophil depletion studies in the murine model have addressed the influence of these cells in host defense However the antibody used in these studies is now known to recognize both neutrophils and inflammatory monocytes therefore the role of neutrophils is still unsettled 28 Although neutropenic patients are at risk for some fungi histoplasmosis is not among them 29 After cellular activation via Th1 cytokines IFNγ and GMCSF macrophages inhibit intracellular growth of H capsulatum 30 The mechanisms deployed by murine and human macrophages are distinct While phagosome acidification is used by murine macrophages it is dispensable within human macrophages 31 H capsulatum phagocytosis stimulates a Horwath et al Page 3 Future Microbiol Author manuscript available in PMC 2016 April 01 Author Manuscript Author Manuscript Author Manuscript Author Manuscript respiratory burst within human but not murine macrophages However studies utilizing NADPH oxidasedeficient mice suggest that inhibition of fungal replication in vivo may require ROS production 32 Additional mechanisms of inhibition suggested by in vitro studies include reactive nitrogen species and metal deprivation 3335 Even with activation macrophages cannot efficiently sterilize tissues and thus harbor H capsulatum 30 Infected macrophages induce granuloma formation in immunocompetent patients 1 36 While granulomas were thought to be a form of host protection recent evidence suggests that Mycobacterium tuberculosisinduced granulomas serve as a repository for pathogenic organisms 37 Reactivation histoplasmosis is a recognized clinical entity particularly in individuals that have left endemic areas into regions that are not known to contain the fungus 38 39 Although studies in the 1950s indicated that healed granulomas only contained dead organisms the efficacy of organism recovery is unknown 40 Thus the source of organisms that cause reactivation remains enigmatic Adaptive immune response Induction of immunity As the most potent antigenpresenting cells DCs provide a link between innate and adaptive immunity Human DCs are capable of killing H capsulatum 25 This fungicidal activity was dependent on lysosomal hydrolases but not the respiratory burst or nitric oxide production Human DCs drive CD4 or CD8 Tcell proliferation by presenting H capsulatum antigen directly or from apoptotic macrophages respectively 25 41 Adoptively transferred H capsulatumloaded DCs are able to suppress maladaptive IL4 production and improve survival following CD4 depletion 42 Thus the DC ability to kill yeast and present antigen in the absence of activating cytokines indicates they are drivers of an effective Tcell response Early studies demonstrated that CD4 and CD8 T cells are necessary for an effective immune response nude mice that lack T cells exhibit high mortality following a low dose H capsulatum infection 43 CD4 Tcell depletion during primary infection led to murine death loss of CD8 T cells decreased clearance efficiency 44 Following vaccination CD8 T cells confer protection while CD4 T cells are dispensable 45 Increased mortality and fungal burden in the absence of T cells is caused by a lack of protective cytokines such as IFNγ and TNFα 44 46 An increased incidence of histoplasmosis in AIDS patients supports the protective role of CD4 T cells in humans CD4 and CD8 T cells influence reactivation elimination of both 6 weeks after infection elevates fungal burden 47 These studies mimic the human situation in which AIDS patients living in nonendemic areas exhibit reactivation disease concomitant with low CD4 Tcell counts 38 The protective effect of antibody that develops after infection is minor at best Bcell knockout KO mice exhibit no change in fungal burden during primary infection but exhibit elevated fungal burden and a prolonged resolution in secondary disease 44 Others have reported that monoclonal antibodies to H capsulatum surface ligands are protective 48 Horwath et al Page 4 Future Microbiol Author manuscript available in PMC 2016 April 01 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Th1 response protagonists in clearance of H capsulatum During infection CD4 T cells polarize into several helper phenotypes such as Th1 Th2 Th17 and Treg with unique cytokine profiles IL12 promotes Th1 differentiation and IFNγ production In murine histoplasmosis IL12 blockade accelerates murine mortality in an IFNγdependent manner 49 In histoplasmosis IFNγ is produced primarily by Th1 cells activates intracellular killing and is necessary for control of primary infection 50 IFNγ also contributes to survival in secondary infection 50 51 Lipid mediators may be part of the Th1 response leukotriene inhibition in murine histoplasmosis results in decreased IFN γ IL12 and survival 52 The importance of IFNγ signaling in humans has been established with reports linking genetic deficiency in the receptor to disseminated disease 53 TNFα another protective Th1 cytokine exerts multiple effects including activation of phagocytic cells induction of apoptosis and control of the CD4 phenotype TNFα neutralization impairs survival of mice 54 In primary infection TNFα blockade reduces nitric oxide production by macrophages By contrast the major defect in secondary infection is overproduction of detrimental IL4 and IL10 54 In both primary and secondary infection TNFα neutralization abolishes the ability of murine T cells to mediate protection from H capsulatum 55 TNFα induces caspase activation additional TNFα production and apoptosis in infected macrophages 56 This result suggests a protective mechanism in which yeast replicating in permissive macrophages are released by apoptosis along with an additional burst of TNFα to neighboring cells Studies in mice regarding the importance of TNFα are confirmed by clinical use of TNFα blockers These medications increase patient susceptibility to H capsulatum and patients are especially at risk for severe disseminated infection 57 GMCSF is another important inflammatory cytokine produced by multiple cell types including Th1 cells GMCSF promotes both differentiation and activation of myeloid cells such as macrophages and neutrophils neutralization leads to murine death following H capsulatum infection 58 GMCSF acts at least in part by promoting fungistatic activity in macrophages through sequestration of zinc a novel mechanism for pathogen control 34 Loss of IFNγ TNFα GMCSF or IL12 can each independently increase murine mortality in histoplasmosis Th17 response supporting characters in H capsulatum control Th17 cells produce inflammatory cytokines such as IL17 IL6 and GMCSF The Th17 response is important for controlling many fungal infections and may be beneficial but not essential for control of H capsulatum In wildtype mice IL17 neutralization results in a larger fungal burden but does not alter resolution of infection 59 Conversely elevated IL17 improves fungal clearance in the CCR5 KO mouse 60 In humans HyperIgE syndrome HIES is defined by a mutation in STAT3 leading to decreased Th17 cells Even though HIES is a rare disorder there are several case reports of HIES patients that developed disseminated or gastrointestinal histoplasmosis 61 The fact that the gastrointestinal tissue is a target in most of these patients suggests that as with mucosal Horwath et al Page 5 Future Microbiol Author manuscript available in PMC 2016 April 01 Author Manuscript Author Manuscript Author Manuscript Author Manuscript candidiasis Th17 cells may be more important in the regulation of mucosal rather than systemic immunity for H capsulatum Th2 Treg villains in histoplasmosis In contrast to Th1 and Th17 cytokines representative of Th2 responses exacerbate histoplasmosis Th1 and Th2 responses are mutually antagonistic Th2associated cytokines polarize macrophages to a M2 phenotype and these cells assist in tissue repair but fail to kill various intracellular pathogens 62 IL4 is a critical type 2 cytokine and transgenic mice overexpressing IL4 exhibit delayed fungal clearance 63 CCR2 KO mice also exhibit increased IL4 production and impaired clearance 64 In these mice IL4 triggers production of another Th2associated cytokine IL33 by infected macrophages 65 Subsequent to these elevated cytokines a shift to the maladaptive M2 macrophage phenotype occurs Neutralization of either IL4 or IL33 improves H capsulatum clearance in this model Tregs mediate immune suppression in several ways including cellcell interactions and production of cytokines such as IL10 and TGFβ While important for limiting excessive immune activation IL10 impedes clearance of H capsulatum 66 This cytokine negatively affects development of the protective Th1 response in histoplasmosis IL10 KO mice develop more IFNγ CD4 cells and clear the infection more rapidly 66 Treg balance with other Tcell subsets is important CCR5 KO mice exhibit a shift toward Th17 which leads to decreased Treg numbers and IL10 production 60 These mice have accelerated clearance of H capsulatum both the elevation in Th17 and depression of Treg response may improve intracellular killing Since IL17 appears to be dispensable for H capsulatum clearance decreased Tregs are likely responsible for this phenotype Metals immunity in histoplasmosis Deprivation of trace metals is an effective immune strategy to slow or stop an infection In murine histoplasmosis activation of macrophages involves mechanisms that limit at least two essential minerals iron and zinc H capsulatum must obtain iron bound to ferritin or transferrin in the phagosomelysosome compartment It possesses several means to accomplish this including siderophores ferric reductases and maintenance of favorable pH 1315 67 Murine macrophages activated with IFNγ and lipopolysaccharide counteract H capsulatum iron acquisition possibly via NO production conversely increasing iron availability increases yeast survival 33 35 This mechanism of H capsulatum control has not been reported in human macrophages and the importance of iron in clinical histoplasmosis is unresolved Zinc must be obtained by an intracellular invader for survival and growth Both human and murine macrophages activated with GMCSF upregulate metallothioneins small metal binding proteins which reduce intracellular free zinc 34 68 Metallothioneindriven reduction of zinc supports production of ROS by increasing activity of the phagosomal H channel 34 Thus GMCSFs ability to inhibit yeast growth in macrophages may be due to zinc deprivation and enhanced ROS In contrast to GMCSF IL4 increases zinc availability to intracellular yeast 68 Horwath et al Page 6 Future Microbiol Author manuscript available in PMC 2016 April 01 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Translation from mouse to human Importance of CD4 T cells in clinical histoplasmosis Our understanding of the importance of CD4 cells in histoplasmosis has emerged from observations in both mice and humans By 1971 physicians noted that patients with histoplasmosis usually exhibited an increase in T or B lymphocytes while patients with progressive disseminated disease tended to lack this response 43 Early studies in athymic mice confirmed the importance of T cells for control of histoplasmosis 43 In the 1980s the HIV epidemic brought a dramatic increase in histoplasmosis incidence severity and awareness An impaired CD4 response is the major risk factor progressive disseminated histoplasmosis typically presents in those with low CD4 Tcell counts 100 cellsmm3 Today most instances occur in patients not receiving HAART Mortality in this group may approach 50 2 Patients taking medications that suppress CD4 cell number or function are another major population with increased risk for histoplasmosis particularly disseminated disease Glucocorticoids effect all immune cell types but have an especially dramatic influence on Tcell number and function 69 Highdose glucocorticoids either alone or in combination with other immunosuppressive agents have been linked to histoplasmosis 7072 Immunosuppressants that target T cells including folic acid antimetabolites calcineurin inhibitors and monoclonal antibodies targeting lymphocytes are associated with cases of histoplasmosis 7375 Exact assessment of risk is difficult because many patients take multiple medications have an underlying condition that may influence immunity or both TNFα neutralization human disease Our understanding of the mechanism of TNFα in histoplasmosis has evolved through both murine studies and clinical observations The requirement of TNFα for optimal control of histoplasmosis was first observed in mice and the ability of TNFα to suppress detrimental cytokines such as IL4 and IL10 was elucidated in this model 54 These findings predicted the relevance for human disease which was eventually revealed by the introduction of TNF α blocking therapeutics Histoplasmosis is the most commonly reported fungal infection associated with TNFblockers and affected patients are at risk for severe disseminated histoplasmosis 57 76 Clinical studies revealed the ability of TNFα to antagonize Tregs reduced Treg number and function in rheumatoid arthritis patients is restored by TNFα blockers 77 This observation prompted further studies in murine histoplasmosis TNFα blockade in mice was found to induce an expanded pool of H capsulatumspecific suppressive T cells 78 Unexpectedly these T cells did not have typical Treg markers therefore TNFα blockade may also act by promoting suppressor function in nonTreg CD4 populations 78 Recent human studies have reflected this finding showing that TNFα blockers can induce immunosuppressive features in Th17 cells 79 Conclusion Innate immune recognition of H capsulatum by macrophages and dendritic cells is critical for both early cytokine and chemokine production and phagocytosis These innate cells Horwath et al Page 7 Future Microbiol Author manuscript available in PMC 2016 April 01 Author Manuscript Author Manuscript Author Manuscript Author Manuscript express effector cell function and are necessary for promoting differentiation and recruitment of Th1 cells The failure to generate a robust Th1 response results in a collapse of immunity Enhanced generation of Th2 or Treg cells antagonizes the development of protective immunity to this fungus in part by counteracting the action of Th1 cells Clinical evidence supports the importance of Th1 cells and TNFα in host defenses to this pathogen The overlap between murine data and clinical studies suggest that studies in mice are useful for delineating the human immune response toH capsulatumeven in the context of novel immunosuppressive drugs Future perspective Since the murine model seems to accurately reflect the clinical picture murine and clinical studies for H capsulatum should continue to inform each other to advance future research This dialogue has already led to important insights Murine models correctly predicted that individuals taking antiTNFα drugs would have an increased susceptibility to histoplasmosis these models also helped explore the role of an expanded Treg population following treatment Targeted antibody therapies that block specific cytokines or receptors provide powerful new tools that target autoimmune diseases or transplant rejection However these medications put the patient at risk for infection Knowledge from murine studies will help predict risk for histoplasmosis as new therapies are introduced Mavrilimumab the first in a new class of GMCSF blocking therapeutics has shown promising results in Phase I and II clinical trials for treatment of rheumatoid arthritis 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transplant recipients receiving alemtuzumab Transplant Proc 2005 372934936 PubMed 15848579 76 Hage CA Bowyer S Tarvin SE Helper D Kleiman MB Wheat LJ Recognition diagnosis and treatment of histoplasmosis complicating tumor necrosis factor blocker therapy Clin Infect Dis 2010 5018592 PubMed 19951231 77 Ehrenstein MR Evans JG Singh A et al Compromised function of regulatory T cells in rheumatoid arthritis and reversal by antiTNFα therapy J Exp Med 2004 2003277285 PubMed 15280421 78 Deepe GS Gibbons RS TNFα antagonism generates a population of antigenspecific CD4CD25 T cells that inhibit protective immunity in murine histoplasmosis J Immunol 2008 180210881097 PubMed 18178849 Demonstrates generation of immunosuppressive T cells by TNFα inhbition reflecting clinical data 79 Evans HG Roostalu U Walter GJ et al TNFα blockade induces IL10 expression in human CD4 T cells Nat Commun 2014 53199 PubMed 24492460 80 Di Franco M Gerardi MC Lucchino B Conti F Mavrilimumab an evidence based review of its potential in the treatment of rheumatoid arthritis Core Evid 2014 94148 PubMed 24648832 Horwath et al Page 12 Future Microbiol Author manuscript available in PMC 2016 April 01 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Executive summary Histoplasma capsulatum a global fungal pathogen Histoplasmosis typically presents in immunocompromised patients living in endemic regions Strains of Histoplasma capsulatum are phenotypically unique which may alter the infection and immune response H capsulatum yeasts are adapted to infect mammalian macrophages Innate immune response Macrophages and dendritic cells utilize different surface receptors to phagocytose H capsulatum which activate different signaling cascades and may lead to differential response Macrophages must be activated to inhibit H capsulatum survival Granulomas represent a potential source of pathogens for reactivation disease Adaptive immune response Antigen presentation and induction of H capsulatumspecific CD4 Tcell response is essential for clearance of infection Protection requires polarization of the CD4 Tcell response for production of protective Th1associated cytokines and suppression of detrimental Th2 and Tregassociated cytokines Th17 response may be important in the human response to H capsulatum although it is dispensable in murine disease Protective cytokines trigger iron and zinc sequestration from H capsulatum to facilitate clearance of infection Translation from mouse to human Clinical studies support murine data that CD4 T cells are critical for host protection from histoplasmosis In histoplasmosis TNFα is an essential cytokine first identified in murine models and confirmed by clinical use of TNFα inhibitors Horwath et al Page 13 Future Microbiol Author manuscript available in PMC 2016 April 01 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Histoplasma Capsulatum Mechanisms for Pathogenesis Jamie Mittal Department of Medicine Infectious Diseases Montefiore Medical Center Bronx NY USA Maria G Ponce Department of Medicine Infectious Diseases Montefiore Medical Center Bronx NY USA Inessa Gendlina Department of Medicine Infectious Diseases Albert Einstein College of Medicine Bronx NY USA Joshua D Nosanchuk Department of Microbiology and Immunology Albert Einstein College of Medicine Bronx NY USA Abstract Histoplasmosis caused by the dimorphic environmental fungus Histoplasma capsulatum is a major mycosis on the global stage Acquisition of the fungus by mammalian hosts can be clinically silent or it can lead to lifethreatening systemic disease which can occur in immunologically intact or deficient hosts albeit severe disease is more likely in the setting of compromised cellular immunity H capsulatum yeast cells are highly adapted to the mammalian host as they can effectively survive within intracellular niches in select phagocytic cells Understanding the biological response by both the host and H capsulatum will facilitate improved approaches to prevent andor modify disease This review presents our current understanding of the major pathogenic mechanisms involved in histoplasmosis 1 Introduction Histoplasma capsulatum is an environmental dimorphic fungus Wheat et al 2007 Human infection occurs after the fungus in the form of microconidia or hyphal fragments is inhaled travels through the respiratory system and reaches the alveoli In the alveoli it transforms into a yeast form a process that can occur both inside or outside of phagocytes Deepe et al 2008 H capsulatum infections are primarily acquired and there is no person toperson transmission with the rare exception of organ transplantation Lenhart et al 2004 Once Histoplasma enters the host it must evade immunemediated and intracellular defenses and find a favorable niche for growth and reproduction which may include dissemination and the development of a state of latency within granulomas Host phagocytes play a central role in the pathogenesis of histoplasmosis as they are the vehicles for dissemination spreading initially to the lymph nodes and later to multiple organs Guimarães et al 2006 The pathogens ability to evade inflammatory responses and the joshnosanchukeinsteinyuedu HHS Public Access Author manuscript Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Published in final edited form as Curr Top Microbiol Immunol 2019 422 157191 doi101007822018114 Author Manuscript Author Manuscript Author Manuscript Author Manuscript intensity of the host immune response determine the severity of symptoms and clinical presentation and whether a state of latency develops with the potential for reactivation Huffnagle and Noverr 2008 Casadevall and Pirofski 2003 The processes involved for this pathogen to survive and cause host damage are discussed here and they highlight the complexity of the interactions between the host and pathogen An understanding of pathogenesis provides insight into the clinical manifestations reasons for reactivation and future therapeutic targets 2 History Histoplasma was first described in 1906 by Dr Samuel Taylor Darling His first report was on the autopsy findings of a 27yearold carpenter from Martinique who was working in the Panama Canal Darling 1906 He likened his findings to what had been observed in protozoan infections Dr Darling coined the name H capsulatum when he saw the invasion of histocytelike cells with these encapsulated organisms Histoplasmosis was later referred to as Darlings Disease Subsequently in 1912 the correct description of the organism was determined by pathologist Henrique da RochaLima who recognized it as a fungus Baum and Schwarz 1957 Since then Histoplasma taxonomically has been divided into three groups based on geographic distribution and clinical manifestations var capsulatum which is the most common worldwide var duboisii found in Africa and var farciminosum known to be a horse pathogen To refine phylogenic classifications Kasuga et al performed phylogenetic analyses on 137 individual isolates representing the three original classifications of Histoplasma Using DNA sequence variation eight clades were genetically identified with seven of them representing isolated groups The clades are called North American class 1 North American class 2 Latin American group A Latin American group B Australian Netherlands Eurasian and the African clade Overall seven of these clades represent isolated groups except for the Eurasian clade which originated from the Latin American group A clade Isolates classified in one of the three original categories were intermixed and were found to be present in multiple phylogenetic clades thus making the original nomenclature obsolete Kasuga et al 2003 Kasugas clade classification is used today to identify Histoplasma fungus however new information on cryptic speciation may lead to further refinements of H capsulatum classifications Sepúlveda et al 2017 3 Epidemiology As evident from the clade grouping H capsulatum is distributed throughout the world including Asia Africa Australia North Central and South America Chakrabarti and Slavin 2011 Loulergue et al 2007 McLeod et al 2011 Colombo et al 2011 In North America a higher incidence has been reported in the Ohio and Mississippi River Valleys via identification of a high frequency of positive skin testing Manos et al 1956 In South America there is a predominance of disease in Brazil Ecuador Venezuela Paraguay Uruguay and Argentina Guimarães et al 2006 A possible cause for this difference in overall distribution may be associated with features of the soil in these areas and climate differences Wheat et al 2007 Histoplasmosis is the most prevalent endemic fungal infection in the US It is estimated that 50 million people have latent infection and 500000 new infections estimated annually Nosanchuk and Gacser 2008 Retallack and Woods 1999 Mittal et al Page 2 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Baddley et al 2011 Despite wide distribution there is still a significant underdiagnosis and hence underreporting of this disease in the US This was evident in a multistate epidemiological surveillance analysis of histoplasmosis performed in US from 20112014 Armstrong et al 2018 It was noted that over a 3year period only 3409 histoplasmosis patients were diagnosed in 12 states including Alabama Arkansas Delaware Illinois Indiana Kentucky Michigan Minnesota Mississippi Nebraska Pennsylvania and Wisconsin Of those identified infected individuals most patients were asymptomatic although patients who presented with symptoms had a significant risk of mortality as 7 of hospitalized patients with histoplasmosis died Armstrong et al 2018 Given its prevalence and potential for causing severe disease further study and a deeper understanding of pathogenesis are crucial in development and implementation of diagnostic and treatment strategies with goals of improving patient outcomes and increasing disease awareness 4 Clinical Manifestations There is a broad range of presentations for histoplasmosis Disease signs and symptoms are mediated by the host immune status with immunosuppressed patients being at higher risk for more severe disease Other factors that play a role in the severity of the disease include the virulence of the fungal strain and the amount of inhaled inoculum Knox and Hage 2010 Most often individuals will be asymptomatic after the acquisition of the fungus but 1 will present with symptoms associated with the infection Kauffman 2007 Symptomatic clinical presentations include acute pulmonary disease disseminated disease and chronic pulmonary histoplasmosis Additional forms of disease can occur Wheat et al 2016 41 Acute Histoplasmosis Symptomatic patients usually present within one to 3 weeks from exposure They can progress to develop fever chills dry cough chest pain myalgias and headaches often described as a flulike syndrome There are no specific imaging findings but often mediastinal lymphadenopathy and infiltrates can be seen Nadel et al 2005 Acute disease can manifest as pneumonia less frequently as pericarditis or with rheumatologic syndromes including arthritis arthralgias and erythema nodosum and mediastinal fibrosis particularly in patients with HLAA2 in whom fibrosis is associated with abnormal host inflammatory response Kataria et al 1981 Wheat et al 1983 Rosenthal et al 1983 Davis et al 2001 Peebles et al 2000 Other possible manifestations of the acute histoplasmosis are broncholithiasis and pulmonary nodules occasionally leading to compression syndromes due to their size Arrigoni et al 1971 Goodwin and Snell 1969 Nodules can also represent the residual changes of acute disease and can be seen in radiographs as single or multiple calcified and noncalcified lesions often described as coin lesions that can mimic and cause concern for malignancy Galetta et al 2007 42 Disseminated Histoplasmosis Disseminated disease most often occurs in individuals exposed to H capsulatum who are immunosuppressed but a large inoculum infection can result in severe disease in 29 of individuals without immunological disorders Larrabee et al 1978 The risk for dissemination is particularly increased in patients with HIVAIDs particularly with a CD4 Mittal et al Page 3 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript count less than 200 cells per μl but chronic steroid use and immunosuppression secondary to malignancy are additional risk factors for disease Kauffman 2007 Knox and Hage 2010 Histoplasmosis Risk Prevention Nacher et al 2014 Symptoms of disseminated disease include fever weight loss and respiratory complaints Clinical findings of lymphadenopathy hepatomegaly and splenomegaly together with bone marrow toxicity on laboratory evaluation are associated with disseminated disease Skin and mucosal lesions can also manifest Disseminated disease can involve multiple other organs including endovascular central nervous system gastrointestinal and suprarenal glands Wheat et al 2016 Since symptoms are nonspecific even disseminated disease can at times evade recognition and if not treated can progress to chronic or latent disease 43 Chronic Pulmonary Histoplasmosis Chronic pulmonary disease typically occurs in older individuals with previously injured lung tissues Goodwin et al 1976 It may follow acute pulmonary infection or develop secondary to persistent slowly progressive disease typically a complication of the acute disease With acute disease there is a progression of disease from pulmonary infiltrates to fibrosis and finally cavitation Wheat et al 1984 Apical bullae can also be part of chronic disease Patients present with symptoms of fever weight loss cough and dyspnea Kauffman 2007 Bronchopleural fistulas are a serious complication of chronic disease Goodwin et al 1976 Due to the presence of cavitary lesions in chronic pulmonary histoplasmosis it can be confused with tuberculosis or other mycotic infections and coinfections are possible Wheat et al 2016 5 Diagnosis and Treatment Although an indepth discussion on diagnosis and treatment is beyond the scope of the current work it merits a brief overview There are currently multiple diagnostic modalities that include antigen detection serology pathology and culture that when combined can achieve high sensitivity and specificity Azar and Hage 2017 Culture of Histoplasma is the gold standard for diagnosis but obtaining adequate tissue and fluid samples for culture can be difficult Considering fungal growth patterns it can take more than 2 weeks to observe mycelial growth which can significantly delay diagnosis Positive cultures are more likely to be found in disseminated disease due to the presumed higher fungal burden in the host When histopathology is available finding yeast cells in tissue is consistent with Histoplasma and supports the diagnosis Another important diagnostic method is antigen detection Antigen testing ie detection of Histoplasma polysaccharide by antibodies can be done on any body fluids and tissues depending on the suspected site of infection but is most often tested in the urine Azar and Hage 2017 Nadel et al 2005 Assi et al 2011 Additionally urine and other body fluid antigen levels can be helpful in monitoring treatment response Serology is more useful for chronic and subacute disease Various techniques include immunodiffusion complement fixation enzyme immunoassay and radioimmunoassay Finally molecular methods are not yet FDAapproved but these approaches can nevertheless be helpful due to high specificity and faster processing time Azar and Hage 2017 Mittal et al Page 4 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript The decision to treat is based on the severity of the disease as well as the host immune status Treatment recommendations are largely based on the 2007 update by the Infectious Disease Society of America Wheat et al 2007 and the 2011 American Thoracic Society statement Limper et al 2011 When the disease is classified as mild acute pulmonary histoplasmosis there is no indication for treatment unless symptoms are present for more than 4 weeks or if the patient is immunocompromised in which cases itraconazole is recommended If the disease is classified as moderate then the treatment of choice is itraconazole which should be initiated regardless of the duration of symptoms Other possible therapeutic options include voriconazole and posaconazole For patients identified as having moderately severe to severe acute pulmonary disease the first line of treatment is liposomal amphotericin B followed by itraconazole The use of steroids is recommended at initiation of therapy if a patient is hypoxemic or presents with respiratory distress Unfortunately if host immunosuppression persists then the medication will need to be given lifelong When treating disease with itraconazole serum drug level monitoring should be performed 2 weeks after therapy is started and every 36 months while receiving treatment Wheat et al 2007 Despite having a wide geographic distribution histoplasmosis continues to be under recognized There is a range of disease states discussed above and clinical severity determined to a large extent by the immune status of the host Additionally as new immunotherapies are developed the number of people at risk for disease is expanding To enrich our understanding of disease epidemiology treatment development and prevention modalities further we will further focus on the pathogenesis of this disease 6 Pathogenesis 61 Introduction H capsulatum is a member of the family Ascomycetes KwonChung et al 1974 It has a mold or mycelial form and a yeast morphology Nosanchuk and Gacser 2008 The mycelial phase is primarily present in the environment in nitrogen enriched soil which is the case when it is contaminated with bird or bat droppings Fungal hyphae measure from 125 to 2 μm in diameter and occasionally hyphal forms can be seen during human infection particularly in the setting of endocarditis Hutton et al 1985 Svirbely et al 1985 There are two types of conidia in the mycelial form macroconidia ranging in size from 8 to 15 μm in diameter and microconidia ranging in size from 2 to 5 μm in diameter it is the latter that can effectively be inhaled and travel as far as the host alveoli Fig 1 The mycelialtoyeast phase transition in H capsulatum is primarily induced by changes in temperature The yeast form is ovoid measuring from 2 to 5 μm in diameter The yeast cells reproduce by polar budding giving them their characteristic narrow budding base and the appearance of a bridge between mother and daughter cells Nadel et al 2005 The yeast phase predominates in host tissues with an optimal growth rate at 37 C Maresca and Kobayashi 1989 The yeast is the pathogenic form of H capsulatum causing acute disease or it can become latent able to potentially reemerge in the setting of immunosuppression However H capsulatum has many obstacles to overcome to cause host injury It must bypass mucosal barriers during acute inoculation evade host immune cell responses and find its niche Mittal et al Page 5 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript within host macrophages Table 1 is a summary of the hostpathogen responses to various components of the immune response When H capsulatum successfully enters macrophages it can effectively avoid host effector responses and multiply Within these cells the fungus can disseminate throughout the host organs These processes highlight the complexity of the interaction of the host and Histoplasma and the interconnected nature of the host response 62 Transformation to the Pathogen H capsulatum pathogenesis begins immediately upon contact with the host Upon entry into to the host and the shift to mammalian body temperature the mycelial form ceases to be metabolically active Thereafter shunt pathways mediated by cysteine and sulfhydryl compounds are thought to induce morphogenesis to the yeast form Sacco et al 1983 This transition process usually takes from hours to days Sacco et al 1983 The transition can be inhibited by sulfhydryl blocking agents which lock the fungus in the mycelial phase Significantly using this blocking system treated H capsulatum is unable to cause disease in wellestablished animal models Medoff et al 1987 which highlights the necessity of the transformation into the yeast form for virulence More recent studies have evaluated genes involved in the transition process Nemecek et al evaluated DRK1 dimorphism regulating kinase Silencing of this gene reduced H capsulatum virulence and suppressed additional factors important to pathogenesis such as CBP1 and AGS1 which were no longer expressed Nemecek et al 2006 Nguyen et al demonstrated that RYP1 required for yeast phase growth which is part of the WOPR family of genes was similarly required for effective morphogenesis and virulence Nguyen and Sil 2008 Webster et al identified RYP2 and RYP3 that are part of the Velvet family of genes as necessary for morphogenesis Webster and Sil 2008 While all the functions of each of these factors have not been elucidated Beyhan et al attempted to clarify the interactions between Ryp1 Ryp2 and Ryp3 and identified another transcription factor Ryp4 involved in the transformation process Beyhan et al 2013 Using whole genome transcriptional profiling they observed that 96 of yeast phase transcripts were dependent on the expression of these transcription factor genes which were found to associate and interact with upstream regions and impact expression of genes that controlled not only transition but also virulence and therefore pathogenesis Further studies are needed to identify additional factors involved in controlling transformation into the yeast form as well as characterizing possible pathways for interaction Understanding these processes may lead to the identification of potential therapeutic targets in the future However the transformation from the environmental mycelia morphology to the yeast form is only the beginning of the process of successfully infecting the host 63 Mucosal Barriers At the onset of infection aerosolized microconidia enter host airways and the most common types of diseases described are sinus and lung infections Regarding the former disease state it is unclear if this is just a part of dissemination or the result of the nares being the entryway of the pathogen Elansari et al 2016 Rizzi et al 2006 Symptoms of Histoplasmosis In pulmonary and disseminated disease Histoplasma microconidia bypass initial innate defenses such as nasal and pharyngeal mucus mucociliary clearance and initial antibody Mittal et al Page 6 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript defenses likely due to their small size and the negative pressure present with inhalation As Histoplasma spores travel down the respiratory tract they undergo phase transition to yeast In the alveoli yeast encounter the first of the host defenses that impact their survival 631 Surfactant ProteinsDuring acute infection or initial inoculation microconidia and newly transitioned yeast forms that successfully pass the filtering systems of the upper airways and reach the alveoli encounter pulmonary surfactant proteins Surfactant is a complex fluid that is composed mostly of phospholipids and four proteins SPA SPB SP C and SPD which have different biological functions SPA and SPD are part of the collectin family as they contain a collagenlike region that is part of the structure of the C type lectin domain McCormack et al 2003 These hydrophilic surfactant components play a role in lung immunity Han and Mallampalli 2015 CarretoBinaghi et al 2016 SPA and SPD bind viruses bacteria fungi and parasites through a carbohydrate recognition domain CRD opsonizing the pathogens to enhance phagocytosis and clearance by neutrophils and macrophages Nayak et al 2012 CarretoBinaghi et al 2016 van de Wetering et al 2004 However this is not the primary way in which surfactant impacts the survival of Histoplasma Surfactant proteins have inherent fungicidal properties McCormack et al showed that Histoplasma yeast cells grown in the presence of SPA and SPD are greatly inhibited McCormack et al 2003 This decrease in viability was associated with a calcium dependent surfactant proteinmediated increase in permeability of Histoplasma cells The complete mechanism leading to increased permeability of the yeast cells is not fully understood The authors proposed that calcium binding leads to conformational shifts in CRD which expose hydrophobic proteins that disrupt the yeast cell wall Also SPA deficient mice were more susceptible to infection compared to wild type Interestingly there was a minimal decrease in clearance of the pathogen in these mice and the authors proposed that this may be due to the continued presence of SPD or rapid phagocytosis of Histoplasma by macrophages The growth of the pathogen within macrophages was uninhibited by the presence of SPA and SPD McCormack et al 2003 CarretoBinaghi et al 2016 Hence the primary role of surfactants is to impede further entry of the pathogen into host tissues and cells 64 Developing a Niche Within Host Macrophages Once they reach alveoli H capsulatum yeast encounter cells of innate immune system While shown to interact with various cell types as part of the innate immune response yeast cells establish their niche within alveolar macrophages Histoplasma enters the macrophage via phagocytosis and is mediated by complement receptors as this does not require opsonization of the pathogen This is important as the lungs are a site poor in serum opsonins Le Cabec et al 2002 Utilization of this mechanism of phagocytosis is beneficial to the pathogen as it does not trigger additional fungicidal pathways which aids in the organisms ability to persist within a cell Long et al 2003 Additionally when initially entering the cell the pathogen must further alter its cell wall to avoid recognition by other macrophage receptors Garfoot and Rappleye 2016 Garfoot et al 2016 2017 Once ingested once in the cell cytoplasm H capsulatum is contained within a phagocytic vacuole Long et al 2003 To persist within this vacuole it must evade further protective responses Mittal et al Page 7 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript to an invading organism The ways that it alters normal function include inactivating phagasomal reactive nitrogen and oxygen species prevention of phagosomal acidification and potentially lysosomal fusion Lastly while Histoplasma is contained within the macrophage it must be able to create and transport its own nutrients The pathogen if successful can continue to grow and divide in this host cell and eventually will travel to the nucleus to induce host cell apoptosis so that it can infect other cells and disseminate Pitangui et al 2015 641 Entry into the Macrophage Complement ReceptorMediated Phagocytosis The first step in the interaction between the yeast form and the macrophage is ingestion via complement receptormediated phagocytosis The main receptors on the surface of alveolar macrophages are LFA1 CD11aCD18 CR3 CD11bCD18 and CR4 CD11cCD18 Bullock and Wright 1987 Each of these receptors has a unique β subunit and a common α subunit When the CD18 subunit of these receptors is blocked 5090 of Histoplasma binding is impeded Garfoot and Rappleye 2016 Newman et al 1990 The receptorligand protein on Histoplasmas surface that is primarily involved with internalization is a heat shock protein Hsp Hsp are regulators of protein folding and are upregulated during times of stress Cleare et al 2017 Long et al 2003 Guimarães et al 2011b These proteins are distinguished and named for their molecular weight and each plays a different role in fungal pathogenesis For instance Hsp70 is upregulated during the morphogenic shift from the environmental filamentous form to the pathogenic yeast phase Cleare et al 2017 Leach and Cowen 2013 Hsp60 exists on the cell wall of Histoplasma yeast forms and it is the ligand of the CR3 receptor on host macrophages Interestingly it is unclear what promotes its expression specifically on the yeast form cell wall Long et al 2003 In a study by Guimarães et al other functions of Hsp60 within Histoplasma were evaluated Guimarães et al 2011b Hsp60 interacted with 126 unique fungal proteins and the number of interactions increased with temperature increase suggesting that this protein has a wide breadth of cellular functions Guimarães et al 2011b Significantly it is crucial in the initial steps leading to persistence in the host macrophages This is evident in a study by Gomez et al where vaccination with recombinant Hsp60 effectively protected mice challenged with a lethal inoculum of Histoplasma Gomez et al 1995 A later study by Guimarães et al had a similar finding using passive immunity with monoclonal antibodies to Hsp60 In their inoculated mice they observed that certain Hsp60binding antibodies prolonged survival decreased fungal burden and organ damage and increased Th1type cytokine levels IL2 IL12 TNFα Guimarães et al 2009 The interaction between Hsp60 and CR3 may be the reason that Histoplasma can enter the macrophage without triggering additional inflammatory cascades Long et al 2003 However the interaction of these proteins is not an isolated event in terms of successful entry into the macrophage The organism has additional changes it will need to undergo to further evade the host immune response Mittal et al Page 8 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Alterations in the Cell Wall Macrophages have additional cell wall receptors that can recognize fungalpathogenassociated molecular patterns PAMPs to trigger an attack on an invading pathogen One such receptor is Dectin1 This receptor recognizes βglucan which comprises a large part of the Histoplasma cell wall structure This interaction of the glucan and Dectin1 triggers a proinflammatory response from the macrophage The primary mechanism that H capsulatum utilizes to evade Dectin1 is the production of αlinked glucans that surround and conceal βglucans Garfoot and Rappleye 2016 Garfoot et al 2016 These αglucans are synthesized by α13glucan synthase Ags1 Rappleye et al demonstrated that reduction or loss of αglucans results in a significant reduction in virulence Rappleye et al 2004 Additional genes are involved in the process of αglucan synthesis Marion et al identified an α14amylase Amy1 and the gene responsible for producing this protein AMY1 Marion et al 2006 As a part of the αamylase family of enzymes Amy1 may generate α14 linked oligosaccharides that are then used by Ags1 to generate α13 linked glucans or it may be responsible for transglycosylation of the final product Another gene identified in this study was UGP1 This gene generates a UTP glucose1phosphate uridylltransferase that produces UDPglucose monomers These are used by Ags1 to generate a14 and a16 linked glucans With silencing of UGP there was a decrease in substrate and therefore a loss of α13glucan synthesis Marion et al 2006 Strains that have significant quantities of α13glucan as part of their cells walls are called chemotype 2 strains Some strains of Histoplasma lack αglucan production as an evasive mechanism yet these strains show no difference in their virulence and are called chemotype 1 strains This is true of the Panamanian G186A and North American isolate G217B Edwards et al 2011 In a study by Edwards et al genome analysis of the promoter region of the Ags1 gene in αglucandeficient strains showed a large interrupting sequence that demonstrated a decrease in gene product expression in vitro Edwards et al 2011 However AGS1 mRNA was detected upon lung infection in mice The authors proceeded to generate mutant yeast forms that lacked AGS1 and no defects were seen in their ability to infect the lung and disseminate These strains were also used in the study described above by Rappleye et al Those authors found a decrease in virulence with the loss of AGS1 and they argue that α13glucan is still part of the cell wall of these strains but it may be modified and undetectable by standard methods Rappleye et al 2004 Both studies also propose that chemotype 1 strains may utilize unique mechanisms other than the manipulation of their cell wall structure to evade interaction with Dectin1 Edwards et al 2011 Rappleye et al 2004 It is possible that secreted glucanases may be the unique or predominately utilized mechanism of certain strains to evade additional interactions with macrophage receptors The glucanase Eng1 was studied by Garfoot et al using strains G186A and G217B Garfoot et al 2016 When ENG1 expression was silenced the growth of the organism itself was not impacted However in vivo Eng1 deficiency led to reduced infectivity and increased pro inflammatory marker production Hence Eng1 is a secreted glucanase that reduces the amount of βglucan exposed to the macrophages Garfoot et al 2016 Mittal et al Page 9 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Exg8 is another studied glucanase whose target is also cell wall βglucan Exg8 like Eng1 is only produced by the pathogenic yeast form of Histoplasma A second study by Garfoot et al using strains G186A and G217B found that the loss of Exg8 led to a modest attenuation in contrast to Eng1 loss Garfoot et al 2017 Also Exg8 did not significantly impair yeast cell interaction with Dectin1 Exg8 in this study is characterized as an exoglucanase and Eng1 is an endoglucanase It was proposed then that the structure of cellular βglucans is not simple chains with exposed terminal ends but loops hence the endoglucanase Eng1 is of greater importance in evasion of Dectin1 These glucanases maintain the virulence of strains that lack αglucan production but they do not alter the receptorligand binding ultimately leading to internalization by the macrophage ie CR3 binding to Hsp60 Garfoot et al 2017 642 Challenges to Overcome Once Within the MacrophageThe processes discussed thus far have focused on the initial entry and phagocytosis by macrophages Once inside Histoplasma survives within a phagocytic vacuole in the cytoplasm To persist and grow in this compartment it needs to overcome additional challenges Most of the fungus actions are defensive in nature It must inactivate reactive oxygen and nitrogen species released into the vacuole prevent phagosome acidification and lysosomal fusion and produce and transport needed nutrients to overcome nutritional immunity of the macrophage Inactivating Reactive Oxygen Species Ingestion of the fungus by macrophages triggers an oxidative burst which is a surge in the production of reactive oxygen species by the phagosome membrane associated complex NADPH oxidase Missall et al 2004 This enzyme reduces oxygen to superoxide this is secreted into the phagosome and can cause considerable damage to the microbe within it This attack occurs extracellularly to the organism as superoxide is charged and does not cross the cell membrane of the pathogen Garfoot and Rappleye 2016 Youseff et al 2012 Superoxide is not the only chemical that is potentially fungicidal within the phagosome It can be broken down into hydrogen peroxide or it can combine with nitric oxide and both can cause damage to microbes Missall et al 2004 Hydrogen peroxide can also be further broken down into hydroxyl radicals Hydrogen peroxide unlike superoxide can pass through the cell membrane of an organism since it carries a neutral charge and has the potential to cause damage both extraand intracellularly Youseff et al 2012 Overall the susceptibility of a fungus to generated reactive oxygen species is organism dependent Missall et al 2004 For instance Histoplasma yeast cells endure concentrations of reactive oxygen species that would kill other yeast like Candida Youseff et al 2012 Histoplasma as an intracellular pathogen needs to be able to combat both extracellular and intracellular free radicals while in the phagosome These survival mechanisms are crucial to the virulence of the organism hence production of certain enzymes only occurs within the pathogenic yeast form Garfoot and Rappleye 2016 Youseff et al 2012 Eissenberg et al suggested that H capsulatum may not induce an oxidative burst in all macrophages it invades Garfoot and Rappleye 2016 Eissenberg and Goldman 1987 The mechanism of this early finding has never been fully elucidated One explanation is that Mittal et al Page 10 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript murine peritoneal macrophages were used which do not behave the same as human macrophages Eissenberg and Goldman 1987 Youseff et al 2012 In a 2012 study by Youseff et al using functional assays on murine intraperitoneal macrophages further characterized the process of respiratory burst by macrophages infected with Histoplasma Youseff et al 2012 They first observed that the yeast forms infected inactivated macrophages They then created a strain that lacked superoxide dismutase Sod3 which is needed for clearance of free radicals by the fungus within the phagosome Both the wild type and mutant strains infected inactivated macrophages but the survival of the mutant strain was decreased by a small but significant amount This finding indicates that even in the resting state macrophages continue to produce reactive oxygen species The authors also found that a Histoplasma superoxide dismutase decreased free radical levels to baseline within 1015 min of exposure Youseff et al 2012 Additionally they observed that activation with certain cytokines like IFNγ and TNFα that are released by CD4 cells resulted in a further enhancement in the production of reactive oxygen species by macrophages Youseff et al 2012 This highlights the connections and cooperation between the innate and adaptive immune response that are discussed in further detail below Hydrogen peroxide is produced extracellularly by the phagosome and as a byproduct of superoxide production To combat hydrogen peroxide H capsulatum produces 3 catalases CatA CatB and CatP Johnson et al 2002 CatA is produced by the mycelial form of certain strains and production is inducible in the presence of hydrogen peroxide Holbrook et al 2013 Guimarães et al 2008 CatB and CatP are constantly produced by the yeast form They differ in that CatB exerts its role extracellularly while CatP works intracellularly but both function to reduce hydrogen peroxide into water and oxygen to prevent damage to the microbe Guimarães et al 2008 Holbrook et al 2013 In a study by Holbrook et al the authors evaluated the relative importance of each catalase by studying the impact of their removal in generated mutant strains compared to a wild type Holbrook et al 2013 Loss of CatB had no significant impact on virulence in vivo and only minor effect on survival in culture The proposed explanation is that superoxide may be more abundant and play a more significant role as a fungicidal agent Mutants that lacked both Sod3 and CatB were evaluated and there was no additive effect in decreased survival and virulence in vitro and in vivo with the additional loss of CatB The conclusion made was that CatB and CatP alone are not sufficient to protect the yeast form from damage by generated free radicals and superoxide is the major fungicidal agent in the phagosome However the loss of both catalases in a strain while maintaining Sod3 function was evaluated and their loss resulted in a minor reduction in virulence Loss of CatP or CatB alone did not impact virulence to the same degree as seen when both enzymes are silenced The authors asserted here that there is a redundancy in the function of these enzymes and it is likely that this is a result of the nature of their target molecule Hydrogen peroxide carries no charge and so unlike superoxide it can pass freely through the pathogens membrane Unfortunately wherever it travels it is flanked by a catalase on either side Holbrook et al 2013 Garfoot and Rappleye 2016 The pathogen if successful in neutralizing these reactive oxygen species is another step closer to claiming its niche where it can persist grow within and utilize it to disseminate throughout the host Mittal et al Page 11 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Inactivating Reactive Nitrogen Species As mentioned superoxide can also combine with nitric oxide which generates reactive nitrogen species that the organism must also contend with Garfoot and Rappleye 2016 Chao et al 2008 Lane et al 1994 Nakamura et al 1994 Nittler et al 2005 Activated macrophages utilize the enzyme nitrogen oxide synthase to produce nitric oxide Activation of this enzyme is induced by the initial infection and by IFNγ stimulation of macrophages Garfoot and Rappleye 2016 Lane et al 1994 Nakamura et al 1994 Nitric oxide once generated and transformed into a reactive nitrogen species can cause DNA and membrane damage inhibit cell replication and can inactive crucial cellular enzymes Nittler et al 2005 Reactive nitrogen species in response to infection with Histoplasma leads to a fungistatic not fungicidal result Nittler et al 2005 Garfoot and Rappleye 2016 Nittler et al utilized functional genomics to identify a set of genes that were induced in response to reactive nitrogen species in Histoplasma infection They identified 153 gene transcripts that were upregulated with infection but their function remained unclear and no core group of genes could be identified Nittler et al 2005 One gene found was NOR1 and this was noted to have high sequence homology with other nitric oxide reductases seen in other fungal and bacterial pathogens that facilitate the conversion of nitric oxide to nitrous oxide which is no longer toxic to the cell Nittler et al 2005 Chao et al were able to confirm with cell culture mass spectroscopy and nitrous oxide detection that Nor1 reduced reactive nitrogen species into less toxic substances in the infected macrophage Chao et al 2008 It was also found to have constitutive expression in the mycelial form and inducible expression in the yeast form of Histoplasma Nittler et al 2005 Chao et al 2008 It is evident that H capsulatum has defense mechanisms in place to evade both reactive oxygen and nitrogen species But these are not the only dangers to the pathogen when it exists intracellularly and within a phagocytic vacuole Prevention of Phagosome Acidification and Lysosomal Fusion As a phagosome matures it creates an acidic pH internally to enhance microbicidal activities against encompassed microbes Isaac et al 2013 Vacuolar ATPases VATPase are pumps on the membrane that are used to bring protons to the inside of the phagosome Subsequently the phagosome fuses with a lysosome that contains multiple hydrolases that work best at this generated low pH These enzymes then further attack an invading microbe Therefore for the microbe to persist it must find a way to manipulate its environment within the macrophage to create a more basic pH and potentially prevent lysosomal fusion Strasser et al found that inhibition of macrophage VATPase had no effect on H capsulatum survival and there was no change in internal pH Strasser et al 1999 They also found that there was diminished phagosomelysosomal fusion The authors go on to assert that V ATPase was not required for the acidification of phagosomes containing Histoplasma It was thought also that Histoplasma itself contains a pH sensing ability for it to respond to changes in its environment Strasser et al 1999 Isaac et al utilized genetic screening of insertional mutants of Histoplasma to identify mutants that were unable to lyse host macrophages Isaac et al 2013 They identified HMG CoA lyase HCL 1 as being required for growth within the macrophage and later lysis of the cell particularly in glucosedeficient and leucinerich Mittal et al Page 12 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript environments Indirectly the authors found that this enzyme was also important to maintaining phagolysosomal pH In mutants lacking this enzyme there was an accumulation of acidic species when leucine was utilized as the primary energy source while grown in unbuffered media Hcl1 mutants were able to persist in macrophages but their growth was significantly restricted compared to wildtype strains Isaac et al 2013 Unfortunately in vivo studies with mice models did not show a difference in virulence of the mutant strains compared to the wild type This would also suggest that there are additional pathways that require alteration to inhibit pathogenesis and further studies are needed to explore that Isaac et al 2013 Garfoot and Rappleye 2016 Newman et al further investigated the role of pH in H capsulatum survival in human macrophages Newman et al 2005 They found that intraphagosomal pH was about 65 when viable yeast forms were present but there was no change in pH noted using heatkilled cells fixed yeast cells or nonpathogenic yeast known to cause acidification when digested by macrophages This would indicate that an acidic pH may in fact not be needed for killing The authors asserted that additional studies are needed to further explore this relationship out particularly if there is a target or signal pathway needed to initiate changes in pH that aid in clearance of Histoplasma Newman et al 2006 The prevention of lysosomal fusion may be another method utilized by Histoplasma as part of its pathogenesis P338D1 mice and J7742 cell line models have normal fusion of these intracellular compartments Strasser et al 1999 Taylor et al 1989 Eissenberg et al 1988 However the RAW 2647 cell line and human macrophages show a decrease in phagosome lysosome fusion Newman et al 2006 It remains unclear why this is the case Based on the above studies by Newman and Strasser an acidic pH does not seem to limit nor is it necessary for lysosomal hydrolase function Newman et al 2006 Strasser et al 1999 Additional study in this area would be of value not only to understand how the organism may impact this interaction but for the potential of therapeutic interventions that could increase clearance of H capsulatum Nutrient Essential Metal and Nucleic Acid Acquisition At this point if the pathogen defends itself from reactive oxygen and nitrogen species prevents lysosomal fusion and maintains a basic pH as alluded to earlier there are still certain nutrients it will need to acquire that impact its growth and survival The phagosome is generally nutrientpoor Garfoot and Rappleye 2016 This mechanism of limiting nucleic acids iron and vitamins has also been termed nutritional immunity Woods 2016 Garfoot and Rappleye 2016 It is a dynamic process and one that the macrophage possesses to defend itself Therefore the pathogen needs to be able to utilize what is present or produce its own nutrients to meet its metabolic needs Garfoot and Rappleye 2016 Woods 2016 Iron Histoplasma must find a way to deal with iron deficiency in its environment so it can continue to proliferate while in the phagosome Newman et al investigated the role of intracellular iron and its impact on yeast cell growth Newman et al 1994 Initial cultures of the pathogen with iron chelators suppressed growth in a concentrationdependent pattern and the effect was reversed with the supplementation of iron Chloroquine had a similar effect on growth but it did so by raising endocytic pH and induced human macrophages to Mittal et al Page 13 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript kill yeast cells Its effects were reversed using iron supplementation that was soluble at a basic pH Chloroquines effects were both time and dosedependent and impacted fungal burden and dissemination This study highlighted the importance of iron to Histoplasma survival and begs the question of potential therapeutic options Newman et al 1994 Within the host iron is bound and transported into a cell by surface transferrin molecules Once in the cell the iron is free or can be bound to ferritin This binding to ferritin is upregulated in the setting of infection Lane et al found that IFNγ released by T cells during infection caused a decrease in cell surface transferrin receptors Lane et al 1991 This suggests that cytokine stimulation also plays a role in limiting iron availability within the macrophage Intracellular unbound iron is a potential source of this essential metal for Histoplasma When iron is lacking in its surroundings the yeast cells secrete hydroxamates that function as siderophores or iron chelators Howard et al 2000 Garfoot and Rappleye 2016 Howard et al observed that H capsulatumderived siderophores are detected 4 days after inoculation of the media Expression can be suppressed with increasing concentrations of iron in the environment Also the authors found that there is not just one siderophore but 5 different ones Howard et al 2000 Hwang el al later identified the gene SID1 which produces the enzymes that catalyze the first step in siderophore production Strains deficient in this gene showed significant depression in growth Hwang et al 2008 In addition to the chelating activity of siderophores Timmerman et al observed the utilization of enzymatic reductants by Histoplasma to reduce iron and thus allowed for its uptake Timmerman and Woods 1999 The expression of these enzymes was upregulated when the pathogen was grown in irondeficient conditions These included secreted extracellular glutathionedependent ferric reductase extracellular nonproteinaceous ferric reductants and cell surface ferric reducing agents Subsequently they evaluated the relationship between reductases siderophores and discussed pH changes leading to the release of iron from transferrin It was initially believed that an acidic pH allows an organism to gather essential metals while within the macrophage Isaac et al 2013 Strasser et al 1999 Iron is bound to the transferrin receptors and when the pH falls from 70 to 60 there is a 50 dissociation of iron and even more when the pH falls below 60 Timmerman et al noted the function of extracellular glutathionedependent ferric reductase as well as chelation by siderophores despite a pH of 7 and they questioned the true impact of pH changes on iron acquisition Regardless they proposed a model for iron acquisition that included this process The postulate that siderophores ferric reductants and changes in pH can function separately to pull iron off of molecules like transferrin and transport it into the cell Additionally iron bound to siderophores and iron released by transferrin with pH changes can act as the substrate for ferric reductants and once reduced can also be transported into the cell Timmerman and Woods 2001 The process of iron acquisition is crucial to survival for the pathogen however the exact mechanism is complex and there is more to be learned about the genes and enzymes that are involved Winters et al 2008 Additionally other metals like zinc are implicated in pathogenesis but their role and interaction with the pathogen require further study Garfoot and Rappleye 2016 Dade et al 2016 Mittal et al Page 14 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Nucleic Acids Histoplasma can overcome an iron deficient environment similarly it must be able to adapt to the nucleic acid deficient environment of the macrophage To understand how Histoplasma build their DNA studies have focused on developing auxotrophs that are incapable of their own nucleic acid production Nucleic acid auxotrophs were first derived by Woods et al Woods et al 1998 Woods 2016 The URA5 gene when interrupted in a more recent study by Rappleye et al created uracil auxotrophs that had a decrease in growth unless supplemented with uracil in cell culture Woods et al 1998 Rappleye et al 2004 Additionally in this study adenine auxotrophs were created using gene disruption of ADE2 which showed a decrease in growth in cell culture Woods 2016 Rappleye et al 2004 Supplementation of a pyrimidine was required for continued growth However if the pathway was further damaged such that its synthetic function is compromised even with supplementation there is no growth or proliferation of the pathogen Garfoot and Rappleye 2016 Rappleye et al 2004 Woods et al 1998 So it would seem then that Histoplasma is capable of independent production and potentially transport of nucleic acids when needed Vitamins Histoplasma can synthesize its own essential vitamins while within the macrophage Garfoot and Rappleye 2016 Garfoot et al 2014 Garfoot et al identified vitamin synthesis pathways within the genome and then using a medium lacking in nutrients they were able to demonstrate that Histoplasma can produce all essential vitamins except thiamine Garfoot et al 2014 They proposed that the pathogen may gather this vitamin from the host and have scavenging mechanisms to aid in this process Additionally they studied the impact of riboflavin pantothenate and biotin synthesis on growth and proliferation of the pathogen They disrupted RIB2 PAN6 and BIO2 RIB2 mutants persisted in the lungs but they did not replicate in vivo and a similar decrease in virulence was seen when PAN6 was disrupted RIB2 mutants had only partial restoration of growth with supplementation in the cell media BIO2 gene disruption did not impact the virulence of the organism and it is proposed that there is availability within the host to make up for this induced dysfunction The authors asserted that information from this study explains more about the vitamins that are available in the phagosome and thus suggests potential therapeutic targets Garfoot et al 2014 643 Inducing Apoptosis and DisseminationH capsulatum by utilizing the previously discussed interactions with the host macrophage can thrive in its intracellular niche However for infection to be propagated in the host and disseminate to other organs in the body the pathogen needs to induce apoptosis of or otherwise leave the macrophage and infect subsequent phagocytes This process will also potentially activate components of the adaptive immune response to allow for further control by the host and taking residence within granulomas Pitangui et al described the movement of H capsulatum yeast cells within the macrophage They found that yeast aggregate 5 h after infection around the cell nucleus and this leads to DNA damage and cell death Pitangui et al 2015 Deepe et al further defined how apoptosis occurs and what cytokines are involved in this process It is particularly intricate and they describe the utilization of extrinsic pathways that are mediated by and lead to an increased expression of TNFα and activation of caspases 1 and 3 Additionally there is an increase in IL10 production which is counterintuitive as this is a cytokine that inhibits apoptosis in neighboring cells The authors propose that these two Mittal et al Page 15 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript cytokines produce a net effect that benefits the pathogen There is the initial induction of apoptosis and with the stimulation of neighboring cells with IL10 the yeast can go on to infect phagocytes that will accommodate their survival Deepe and Buesing 2012 The question that arises from this is whether Histoplasma triggers cell death or if this is entirely a passive process that occurs when the fungal burden is too high within the macrophage Calcium Binding Protein Cbp1 is specifically produced by H capsulatum yeast cells and it is involved in promoting cell growth in calcium limited settings Batanghari et al 1998 However it also plays a role in cell death and proliferation Sebghati et al 2000 Isaac et al evaluated the mechanisms used by Cbp1 when involved with cell death Isaac et al 2015 The authors screened 14000 insertional mutants to find those that grew at high levels intracellularly but would not lyse cells and three such mutants were identified all lacking CBP1 expression Even with sufficient growth without CBP1 there was no cell lysis indicating that his process was actively induced as opposed to a passive cell death by the pathogen Using whole genome sequencing CBP1 was found to be required for induction of stressresponsive genes that modulate cell death as well as activate caspases 3 and 7 Isaac et al 2015 This is further confirmed in a recent study by English et al which suggests that there is an integrated stress response regulated by Cbp1 that induces the expression of other proapoptotic genes like CHOP and TRIB3 however the entire mechanism by which Cbp1 induces cell death is not known and is an area of further research and potential drug targeting English et al 2017 65 Host Cellular Immune Response Although the macrophage is a principal player in the hostpathogen interaction H capsulatum can interact with a variety of cells before it reaches its niche within the macrophage Deepe et al 2008 Deepe et al demonstrated that the yeast cells preferentially invade different phagocytic cell subpopulations at different times after initial infection They demonstrated that the yeast cells were present in neutrophils dendritic cells DCs and macrophages from days 1 to 7 after inoculation however DCs contained proportionately more yeast cells by day 1 with this shifting towards neutrophils and then macrophages on subsequent days Deepe et al 2008 DCs neutrophils and natural killer cells can effectively kill H capsulatum yeast cells and thus their hostpathogen interaction is distinct from that described so far for macrophages 651 Dendritic CellsDCs first encounter H capsulatum in the alveoli and they serve as a link to subsequent host defenses DCs precursors originate in the bone marrow and mature into more specialized cells that are found in the skin and most solid organs including the lungs specifically in airway epithelium parenchyma submucosa alveolar septal wall and alveolar surfaces Thind et al 2015 Sertl et al 1986 Holt and SchonHegrad 1987 These cells are the primary antigen presenting cells of the innate immune system and serve as a connection to the adaptive immune response as they interact with T cells once they leave the tissues and travel to the lymph nodes Clark and Kupper 2005 Immature DCs engulf entire organisms via receptor or nonreceptor medicated phagocytosis at the tissue level These cells process the pathogen and mature to then become antigen presenting cells Thind et al 2015 Also once activated DCs release cytokines like IL12 and TNFα to Mittal et al Page 16 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript recruit other DC additional phagocyte populations stimulate the adaptive immune response and aid in granuloma formation Zhou et al 2001 DCs efficiently engage H capsulatum Gildea et al observed that after 6 h of incubation 75 of DCs in culture had ingested at least one yeast cell Gildea et al 2001 DCs binding of Histoplasma occurs via a fibronectin receptor on the DC surface called very late antigen5 VLA5 Gildea et al 2001 This was an unexpected finding as there are higher levels of CD18 on the cells surface The authors initially hypothesized that CD18 would be utilized as was the case with macrophages They also observed that DCs inhibited the growth of and killed phagocytized yeast cells unlike what had been observed in macrophages This is similar to what was observed when microconidia are ingested by DCs as their transformation into yeast cells was inhibited Newman et al 2011 The authors proposed that the difference in the receptor interactions of these cells with Histoplasma may impact the intracellular survival of the pathogen although the mechanism for the preferential receptor binding is unknown Gildea et al 2001 Subsequently Gomez et al identified the ligand for VLA5 on the surface of H capsulatum yeast cells which turned out to be a 20 kDa protein called cyclophilin A Gomez et al 2008 Once phagocytosis occurs DCs can be both fungistatic and fungicidal Human DCs phagolysosomal fusion occurs unimpeded Gildea et al 2005 Once inside this vesicle there is either restriction of growth or killing of the organism which largely occurs through hydrolases Nitric oxide and oxygen free radicals do not seem to play a role in DC fungicidal activity as in macrophages Thind et al 2015 Gildea et al 2005 Despite the effective protection provided by the DCs pathogens that are not phagocytized will encounter additional host defenses that can still impact their survival 652 NeutrophilsIn addition to early interactions with DCs after acquisition of H capsulatum the fungus also encounters neutrophils Deepe et al 2008 These cells rapidly arrive at the site of infection to engage the pathogen as part of the innate immune response Thind et al 2015 Human neutrophils are fungistatic against H capsulatum Antimicrobial proteins in neutrophils are contained in azurophilic granules Newman et al 1993 Within these granules there are two families of proteins known as defensins and serprocidins and two additional proteins with unique structures lysozyme and bactericidalpermeability increasing protein BPI Their interactions were rigorously evaluated by Newman et al Newman et al 2000 Defensins HNP1 HNP2 and HNP3 are derived from 29 to 30 amino acids differing only in a single Nterminal amino acid Each of these when incubated with yeast cells showed concentrationdependent inhibition of growth Of the three HNP2 had the greatest inhibitory activity Their effect was also noted to be additive Newman et al 2000 Serprocidins are a group of four proteins Cathepsin G is one of these proteins it is a neutral protease of molecular mass 2931 kDa and it is the only member of this family that inhibits growth of Histoplasma Alone this protein has inhibitory activity and its effects are additive when associated with defensins Newman et al 2000 BPI is a protein within the granule with significant fungistatic ability toward H capsulatum It has a molecular mass of 5060 kDa characterized by a lysinerich aminoterminal and carboxyterminal regions Gray et al Mittal et al Page 17 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript 1989 This protein inhibits the growth of yeast cells in a concentrationdependent manner and it has an additive effect when combined with either defensins or cathepsin G Newman et al 2000 The entire mechanism of fungistasis by these proteins has not been fully elucidated Newman et al 1993 2000 Newman et al have described the discrepancy noted in Histoplasma endemic areas where many individuals who presumably have been exposed to the fungi at some point had negative skin tests when exposed to histoplasmin It is hypothesized that the initial response from neutrophils may be sufficient to clear the organism from the host without the need or time for activation of the adaptive immune response more likely in the setting of a small inoculum Newman et al 1993 Both neutrophils and DC directly impact survival of Histoplasma DCs through cytokine release will impact later control of the infection as well Natural killer cells are also part of the initial host defenses and they are connected to this process as they are recruited by DCs and macrophages 653 Natural Killer CellsIn a study of intranasally infected mice Cain et al examined the inflammatory reactions and cytokine responses with active disease progression Cain and Deepe 1998 Increased levels of IL12 were observed by day 3 of infection followed by the increased expression of IL2 and IFNγ starting on day 5 till day 10 All of these declined as of day 14 It was observed that myeloid cells had increases in their expression by day 5 and by day 7 they peak with the additional presence of natural killer cells It is by day 10 that T cells and B cells start to predominate among the inflammatory cell types at sites of disease This highlights the involvement of natural killer cells in response to infection and a potential connection between the initial myeloid cell response via stimulating cytokines Cohen et al attempted to further define the connections between myeloid cells and the natural killer cells response to infection with various fungal pathogens Cohen et al 2011 Natural killer cell responses were reduced in the presence of H capsulatum yeast cells and associated DCs were unable to produce IL12 Additionally when Histoplasma cells were grown with DCs lacking Dectin1 on the cell surface there was also a reduced natural killer cell response The authors propose a general mechanism for natural killer cells function during a systemic fungal infection They postulate that antigen presenting cells like DC or macrophages produce IL12 in response to fungal cell wall components leading to natural killer cell activation Also their data showed that natural killer cells enhance the production of IL12 from antigen presenting cells as a positive feedback loop Cohen et al 2011 Natural killer cells akin to DCs are directly cytotoxic to the target pathogen however this mechanism is not fully understood regarding infection with Histoplasma Tewari and Von Behren 2000 Natural killer cells also like DCs continue to link the innate and adaptive host response to this pathogen Activated natural killer cells produce IFNγ Zhou et al 2001 which leads to activation of CD8 and CD4 T cells for further control of the infection and potentially granuloma formation Mittal et al Page 18 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript 66 Adaptive Immune Response Granuloma Formation and Reactivation The pathogen at this point has now bypassed the mucosal level obstacles cellular host defenses and is manipulating the macrophage to further disseminate But as alluded to earlier there is a complex network of cytokine production and the adaptive immune responses that are the hosts last line of defense The adaptive immune response once effectively activated can either clear the organism or lead to granuloma formation If the latter occurs there is the potential for reactivation of the organism Reactivation is a response to impaired immunity Allen and Deepe 2006 This can occur in the setting iatrogenic immunosuppression with the use of immunomodulating therapy that impacts cytokine function in conditions like HIVAIDS where there is a loss of T cell function and with additional systemic conditions that impact the immune response Heninger et al 2006 Ultimately the pathogen though potentially controlled after acquisition seems to never truly be eliminated by host defenses The adaptive immune response and granuloma formation is complex and not completely understood but murine models have been developed to examine this process further Allen and Deepe 2006 Heninger et al 2006 As mentioned earlier DC act as antigen presenting cells in lymphoid tissue and activate T cells therefore they are a link between the innate and adaptive immune response Natural killer cells and macrophages serve to connect these responses via cytokine production leading to cell activation Heninger et al used a murine model to further characterize granuloma formation Heninger et al 2006 On day 5 after infection macrophages are present at the tissue and the tissue has a vasculitic appearance that may indicate extravasation of immune cells Granuloma formation occurred in the liver by day 7 The liver granulomas formed were noted to grow and reach their maximum size by day 10 IL10 and TGFβ were elevated early in granuloma formation with the latter coming from infected macrophages The liver granulomas decreased in size after day 10 as immune stimulation waned but pulmonary granulomas did not diminish in size Hence the organ itself has an impact on granuloma characteristics and control responses to the pathogen Heninger et al 2006 Notably 70 of the liver granuloma was made up of macrophages and there was an abundance of IFNγ Subsequently these macrophages produced TNFα DCs and neutrophils were present to a lesser extent as were CD4 and CD8 T Cells Early on there were more CD4 cells but their ratio equalized as time progressed Additionally there was a low level of B cells present within the granulomas which may be a potential link to the formation of protective antibodies Moreover there was a diversity of T and B cells present indicating a diversity in recruitment processes as opposed to single cell type entry multiplication and granuloma formation Heninger et al 2006 Allen et al further elucidated the importance of T and B cells in the granuloma and their role in the control of disease Allen and Deepe 2006 The authors intranasally infected mice and then depleted them of CD4 and CD8 T cells 42 days later The mice developed persistent disease when both cell types were eliminated Also latently infected mice subjected to B cell and CD4 cell depletion resulted in disease reactivation These findings demonstrate that there is cooperation between CD8 T cells and B cells and that this potentially explains why the depletion of either CD4 or CD8 T cells alone was insufficient to Mittal et al Page 19 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript permit reactivation The role of B cells in the immune response is complicated and has yet to be fully defined Tristão et al found that Histoplasma cellfree antigens CFAg are present during murine infection and antibodies were generated against these antigens These antigens competed with actual fungal surface antigens in antibody formation and recognition So as with other components of the immune response this pathogen may have escape mechanisms against antibodies as well Tristão et al 2012 However disease modifying monoclonal antibodies mAb have been described and include antibodies to cell surface displayed histone 2B Nosanchuk et al 2003 Shi et al 2008 M antigen Guimarães et al 2008 Nosanchuk et al 2012 and heat shock protein 60 Guimarães et al 2009 2011a b However there are antibodies that do not improve disease outcomes such as a mAb to H1C Lopes et al 2010 and some mAb can enhance disease such as an IgG2b isotype to heat shock 60 Guimarães et al 2009 Hence the role of antibody therapy in histoplasmosis is complex and requires further investigation The Heninger study found that IFNγ within these granulomas was produced mostly by CD4 and CD8 T cells Heninger et al 2006 As discussed this cytokine activates macrophages to induce killing via reactive oxygen and nitrogen species The macrophage will also produce TNFa To stress the importance of these cytokines in the control of disease there have been studies and case series that evaluate the loss of these cytokines and how that impacts disease progression and reactivation Clemons et al treated rodent models with IFN antibodies and developed IFN knockout mice and compared these to controls Depletion of the cytokine and gene disruption both resulted in a loss of resistance to lethal infection and early mortality in the mice Clemons et al 2000 In a subsequent study by Clemons et al IFN was evaluated as an adjuvant therapy to amphotericin B in infected mice and the combination of the cytokine with amphotericin was superior to drug therapy alone Clemons et al 2001 This experimental work is supported by a case report from Zerbe et al where a patient with an inherited IFNγ receptor deficiency developed recurrent disseminated histoplasmosis Zerbe and Holland 2005 TNFα is also a key regulator of disease Deepe 2005 It is clinically relevant as seen in the study by Lee et al where the authors reviewed postlicensure adverse effects of TNF inhibitors and found 10 cases of reactivation of Histoplasma after treatment with these agents Lee et al 2002 In mouse models TNF depletion or inhibition leads to higher mortality in both primary and secondary infection Allendoerfer and Deepe 1998 Deepe 2005 Granulomas continue to form within the tissues with an increase in inflammatory changes seen specifically in the lungs likely due to an increase in fungal burden Allendoerfer and Deepe 1998 Additionally in primary infection without TNFα there is a decrease in nitric oxide production and this may lead to poor clearance of the organism Deepe 2007 Unexpectedly no change to IFN levels has been observed in this setting which indicates independent production and a possible codependent relationship with TNF α as both are required for clearance of the organism Allendoerfer and Deepe 1998 Deepe 2005 In secondary infection without TNFα there is an upregulation of IL4 and IL10 which diminish protective immunity In fact blockade of both cytokines in TNFα depleted mice improved survival Allendoerfer and Deepe 1998 Deepe 2007 More recent studies are evaluating the T cells populations recruited when TNFα is missing and how this may impact potential therapeutic interventions Deepe and Gibbons 2008 Kroetz and Deepe Mittal et al Page 20 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript 2012 Ultimately further study is required to understand the relationships between this cytokine and the various immune cell responses in mice and eventually expansion to human models to see if these same changes exist It is evident that the interactions of the adaptive and innate immune response eventual granuloma formation and potential reactivation are each complex and interconnected connected processes However further characterization and definitions of the elements involved are beneficial as this can lead to therapeutic targets against disease An example of this is evaluated in a study by LazarMolnar et al as they examined the receptors involved in apoptosis LázárMolnár et al 2008 Programmed cell death1 receptor PD1 is an immune inhibitory receptor that is part of the CD28B7 family It is expressed on activated T cells B cells and myeloid cells PD1 receptor and ligand binding inhibit cytokine production in vitro In PD1deficient mice there is protection from Histoplasma capsulatum infection In wildtype mice blockade of the PD1 pathway generated increased survival by 70 This is a potential pathway to be manipulated as a future therapeutic target Lázár Molnár et al 2008 67 New Pathogenic Mechanisms Extracellular VesiclesExtracellular vesicles are lipid bilayered structures that contain lipids phospholipids polysaccharides nucleic acid proteins and other compounds Extracellular vesicles have been described in all biological kingdoms ZamithMiranda et al 2018 In fungi extracellular vesicles have been shown to transport diverse compounds that include factors associated with virulence Joffe et al 2016 Rodrigues et al 2008 H capsulatum yeast cells produce extracellular vesicles that carry virulence factors such as heat shock protein 60 catalases laccases and phosphatases Albuquerque et al 2008 Matos Baltazar et al 2016 Fungal extracellular vesicles can modulate hostpathogen interactions ZamithMiranda et al 2018 Vargas et al 2015 hence H capsulatum extracellular vesicles are postulated to impact pathogenesis This is supported by the fact that the contents of H capsulatum vesicles are recognized by immune human sera Albuquerque et al 2008 Additionally binding of mAb to H capsulatum heat shock protein 60 to yeast cells induces a change in extracellular cargo loading and the characteristics of released vesicles are distinct from those isolated from untreated yeast cells Matos Baltazar et al 2016 which suggests that this process is dynamic and there is an interplay between the fungus and the host immune system Targeting processes associated with the loading and release of extracellular vesicles is a promising potential approach to modifying the virulence of the fungus 7 Concluding Remarks H capsulatum is the most prevalent endemic fungus worldwide Nevertheless histoplasmosis remains underrecognized and it is thus underreported Disease caused by H capsulatum has a wide range of presentations and varying degrees of severity of clinical manifestations that impact treatment decisions The process of infection and damage to the host is complex and involves a dynamic interaction between host defense mechanisms both at the mucosal and cellular level and the pathogens evasive and reactive responses If the pathogen is successful it can cause acute 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for primary immunity to Histoplasma capsulatum J Immunol 166319681974 PubMed 11160245 Mittal et al Page 29 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Fig 1 A depiction of the acquisition process of mycelial Histoplasma from the environment to the lung with the transformation to the yeast form upon deposition in alveoli Mittal et al Page 30 Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Author Manuscript Author Manuscript Author Manuscript Author Manuscript Mittal et al Page 31 Table 1 Summary of the immune system components their response to an invading pathogen and Histoplasmas protective mechanisms Host mechanisms Pathogen response Referencesa Host entry None Phase transition from mycelial to yeast form Sacco et al 1983 Medoff et al 1987 Nemecek et al 2006 Innate defense Mucociliary clearance mucus production None needed Elansari et al 2016 Rizzi et al 2006 Symptoms of Histoplasmosis Surfactant Pathogen opsonization Alters Histoplasma yeast permeabilitySurfactant proteins A and D SPA and SPD Rapid entry into macrophage intracellular localization shields from the action of pulmonary surfactants McCormack et al 2003 Han and Mallampalli 2015 CarretoBinaghi et al 2016 Macrophages Detects fungal cell components and initiate proinflammatory response Attacks phagocytized pathogen with reactive oxygen and nitrogen species Creates an acidic environment in the phagosome which allows for activation of hydrolases upon lysosomal fusion Creates nutritionally deficient environment Attract other immune cells Avoids immune response on entry Inactivates reactive oxygen and nitrogen species Prevents acidification and lysosomal fusion Produces its own nutrients or has mechanisms for transport to obtain them from the environment Induce apoptosis to further disseminate Long et al 2003 Guimarães et al 2011b Garfoot et al 2017 Missall et al 2004 Newman et al 1994 Dendritic cells Cytotoxic to the pathogen Acts as an antigen presenting cell and secretes cytokines to further propagate the immune response No active defense to this cell Clark and Kupper 2005 Zhou et al 2001 Gildea et al 2001 Neutrophils Traps the yeast forms intracellularly in a fungistatic response using enzymes contained within azurophilic granules to impede growth No active defense to this cell Newman et al 1993 2000 Gray et al 1989 Natural killer cells Cytotoxic to the pathogen Acts as an antigen presenting cells and secretes cytokines to further propagate the immune response No active defense to this cell Cain and Deepe 1998 Cohen et al 2011 Tewari and Von Behren 2000 Adaptive immunity Cytotoxic to pathogen Further propagate the immune response to eventually trap the pathogen within a granuloma Utilizes macrophages as a sanctuary Reactivates with changes to the hosts immune status Allen and Deepe 2006 Heninger et al 2006 Tristão et al 2012 Shi et al 2008 Nosanchuk et al 2003 aSample references provided please see text for full list of references and details on each topic Curr Top Microbiol Immunol Author manuscript available in PMC 2020 May 11 Send Orders for Reprints to reprintsbenthamscienceae 224 The Open Microbiology Journal 2017 11 224282 1874285817 2017 Bentham Open The Open Microbiology Journal Content list available at wwwbenthamopencomTOMICROJ DOI 1021741874285801711010224 REVIEW ARTICLE Paracoccidioidomycosis Current Perspectives from Brazil Rinaldo Poncio Mendes 1 Ricardo de Souza Cavalcante 1 Sílvio Alencar Marques 2 Mariângela Esther Alencar Marques 3 James Venturini 4 Tatiane Fernanda Sylvestre 1 Anamaria Mello Miranda Paniago 5 Ana Carla Pereira 6 Julhiany de Fátima da Silva 1 Alexandre Todorovic Fabro 7 Sandra de Moraes Gimenes Bosco 8 Eduardo Bagagli 8 Rosane Christine Hahn 9 and Adriele Dandara Levorato 1 1Department of Tropical Diseases Faculdade de Medicina de Botucatu São Paulo State University UNESP São Paulo Brazil 2Department of Dermatology Faculdade de Medicina de Botucatu São Paulo State University UNESP São Paulo Brazil 3Department of Pathology Faculdade de Medicina de Botucatu São Paulo State University UNESP São Paulo Brazil 4Laboratory of Experimental Immunology Department of Biological Science Faculty of Science São Paulo State University UNESP São Paulo Brazil 5Department of Infectious and Parasitic Diseases Faculdade de Medicina Federal University of Mato Grosso do Sul UFMS Brazil 6Instituto Lauro de Souza Lima São Paulo State Bauru Brazil 7Unit of Experimental Research Faculdade de Medicina de Botucatu São Paulo State University UNESP São Paulo Brazil 8Department of Microbiology and Immunology Instituto de Biociências de Botucatu São Paulo State University UNESP São Paulo Brazil 9Laboratory of Investigation and Mycology Federal University of Mato Grosso Faculty of Medicine Cuiabá Mato Grosso Brazil Received July 12 2017 Revised October 10 2017 Accepted October 10 2017 Abstract Background This review article summarizes and updates the knowledge on paracoccidioidomycosis P lutzii and the cryptic species of P brasiliensis and their geographical distribution in Latin America explaining the difficulties observed in the serological diagnosis Objectives Emphasis has been placed on some genetic factors as predisposing condition for paracoccidioidomycosis Veterinary aspects were focused showing the wide distribution of infection among animals The cellmediated immunity was better characterized incorporating the recent findings Methods Serological methods for diagnosis were also compared for their parameters of accuracy including the analysis of relapse Address correspondence to this author at the Department of Tropical Diseases Faculdade de Medicina de Botucatu São Paulo State University UNESP São Paulo Brazil Tel 551438801742 Email tietemendesterracombr Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 225 Results Clinical forms have been better classified in order to include the pictures less frequently observesiod Conclusion Itraconazole and the trimethoprimsulfamethoxazole combination was compared regarding efficacy effectiveness and safety demonstrating that azole should be the first choice in the treatment of paracoccidioidomycosis Keywords Paracoccidioidmycosis Paracoccidioides brasiliensis Paracoccidioides lutzii Endemic mycosis Systemic mycosis Antifungal compounds 1 INTRODUCTION 11 Definition Paracoccidioidomycosis PCM is a systemic granulomatous disease that can affect any organ in the body predominantly the lungs organs rich in mononuclear phagocyte system cells the mucous membrane of the upper aerodigestive tract UADT the skin and adrenal glands This condition is caused by thermally dimorphic fungi of the Paracoccidioides brasiliensis complex P brasiliensis P lutzii Pb01 and Pb01like species It is an endemic disease limited to Latin America from Mexico to Argentina As the recognition of P lutzii is recent most of knowledge about the fungus and its interaction with the host has been based on studies with P brasiliensis For this reason this article brings a greater number of references related to P brasiliensis 12 Brief History The two earliest cases of PCM were reported in 1908 by Adolpho Lutz 1 who described the clinical manifestations and anatomopathological findings of the disease and isolated its aetiological agent in pure cultures Lutz also infected guinea pigs observed the occurrence of thermal dimorphism a yeastlike phase in tissues and a filamentous phase in culture media and reproduction by multiple budding Lutz named the disease pseudococcidial hyphoblastomycosis to distinguish it from coccidioidomycosis which is caused by Coccidioides spp as well as from Gilchrists disease currently known as blastomycosis and caused by Blastomyces dermatitidis Despite his major contribution to the understanding of PCM Lutz did not suggest a name for its aetiological agent In 1912 Splendore classified the organism as yeast from genus Zymonema 2 In 1928 Almeida and Lacaz introduced the name Paracoccidioides and Almeida named the fungus Paracoccidioides brasiliensis in 1930 3 Although the disease was given countless names the one most widely employed to identify Lutzs mycosis was South American blastomycosis However reports of autochthonous cases from Central America and Mexico showed that it was not restricted to South America and together with the trend to integrate the name of the disease with the name of its aetiological agent Paracoccidioides brasiliensis led to the preferential use of the term paracoccidioidomycosis which was suggested by Jordan in 1946 4 and officialised at the Medellin Symposium Colombia 5 Reports of many cases of PCM presenting with lesions in the mucous membrane of the UADT led to consideration of this area as the entry point for P brasiliensis into the body However in 1956 GonzalezOchoa suggested that the lungs are actually the entry point 6 a hypothesis that was reinforced by Mackinnons findings in an experimental model 7 The existence of a PCM primary complex was subsequently confirmed by Severo et al 8 The existence of many individuals with Paracoccidioides infection was revealed by Fonseca Filho and Arêa Leão 9 through an intradermal reaction induced using a P brasiliensis culture filtrate as antigen This antigen was termed paracoccidioidin 10 Considering the lungs as the portal of entry for P brasiliensis into the organism the fungus could be isolated in the saprophyte state from nature and could live inside a heterothermic organism native to endemic areas 11 Indeed isolation from the soil was achieved by Albornoz 12 and from armadillos by Naiff et al 13 The histopathological characteristics of PCM were thoroughly investigated by Cunha Motta in patients with lesions affecting organs that are rich in mononuclear phagocyte system cells 14 In turn Fialho 15 demonstrated that lung involvement was very frequent and made an accurate characterisation of it The correlation between histopathological findings and cellmediated and humoral immunity was established at the School of Medicine of Botucatu 16 226 The Open Microbiology Journal 2017 Volume 11 Mendes et al P brasiliensis exhibits a complex antigenic structure that includes glycoproteins glycopeptides lipids and polysaccharides The correlation between virulence and presence of α13glucan in the cell wall was the point of departure for various studies of the biochemistry and dimorphism of the fungus 17 Arc E detected by Yazarbal via immunoelectrophoresis 18 revealed the presence of specific serum antibodies against the 43kDa glycoprotein This protein constitutes the dominant antigen of P brasiliensis and was later characterised by Puccia et al 19 The serological assessment of patients with PCM was first performed by Moses 20 using the complement fixation and precipitation tests which were later standardised by FavaNetto using a polysaccharide antigen 21 22 Next Restrepo introduced the double agar gel immunodiffusion test DID This test was found to be simple to perform to be highly specific and to be useful for the followup of patients undergoing treatment 23 Subsequently Biagione et al 24 found a correlation between the serum levels of antibodies on the DID test and PCM severity The in vitro conversion of the mycelial to the yeastlike phase which confirmed Lutzs original observation mycelial phase in vitro and yeastlike phase in guinea pigs was demonstrated by Negroni 25 and was introduced into the laboratory routine for the identification of P brasiliensis Fluoresceinlinked immunoglobulin conjugates were also added to the techniques used for the identification of P brasiliensis in clinical samples 26 The depression of the cellmediated immune response in patients with PCM was demonstrated by Mendes Rafael 27 and Musatti et al 28 This effect was followed by reports that indicated a correlation between depression of cell mediated immunity and patient severity 29 and that immunosuppression is antigendependent 30 In PCM the various possible outcomes of the hostparasite interaction infection only mild moderate or severe clinical forms as well as hormonal influences point to the relevance of the genetic background for the development of disease The line of research developed by Calich et al with isogenic mice that were susceptible and resistant to Paracoccidioides infection 31 has greatly contributed to the understanding of PCM immunopathology In 1940 the use of sulphapyridine by Oliveira Ribeiro was found to be an efficacious drug for the treatment of PCM 32 The second therapeutic agent amphotericin B an antifungal from another chemical class was introduced only 18 years later by Lacaz Sampaio 33 These two medications represented a revolution in the prognosis of PCM Studies on the phylogeny 34 and genomics 35 of PCMcausing fungi allowed the demonstration of more than one species in the genus Paracoccidioides with variation in their geographical distribution Despite involving individuals joined in agribusiness base of the Brazilian economy PCM still remains a neglected disease due to the following characteristics it occurs in poor and rural environments disproportionately affects low income populations perpetuates a vicious cycle of the disease between poverty and inadequate health care does not receive attention from the developed world is outside the purview of the Global Fund and its related programs promotes poverty by causing longlasting sequelae and devastating impacts on individual work productivity and quality of life generally disables rather than kills involves patients who are not able to obtain the drug therapy and finally it affects patients who frequently ask for medical care very late when the disease is at an advanced stage In addition like several neglected tropical diseases PCM has been extensively studied by researchers from developed countries or from renowned research centers in developing countries 36 37 Surveillance and control programs constitute the first step to change this condition but compulsory notification was not implemented in Brazil in spite of the numerous efforts of the Secretaria de Vigilância em Saúde Ministério da Saúde SVSMS 36 This program was developed as an initiative of some states of the federation 36 2 ETIOLOGY 21 Mycology Fungi of the Paracoccidioides genus are thermally dimorphic and can be cultivated as mycelium or yeast cells Cultivated at 25 oC after 15 to 30 days a white colony is observed becoming velvety and brownish By using agar Sabouraud dextrose it is possible to observe septated hyaline hyphae with branches in this culture medium the production of conidia is rare When cultivated in media without carbohydrates but with natural substrates arthroconidia aleuroconida and arthroaleuroconidia present 2 to 5µm in diameter At 37 oC and in human and animal tissues P brasiliensis resembles yeast cells Its growth is slow showing rugged and pleated colonies from 7 to 20 days after inoculation Under direct microscopy the yeastlike cells vary in size and shape being oval spherical or eliptical with birefringent walls The mothercells present 20 to 30µm in diameter and can produce 10 to 12 daughtercells with 2 to Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 227 10µm forming the characteristic pilot wheel The mothercell with two daughtercells frequently resembles the mickey mouse Figs 1A B D Fig 1 Paracoccidioides brasiliensis A a 10 KOH preparation of sputum showing multiple budding yeast forms with birefringent walls B cellblock preparation of sputum GMS x400 showing the mickeymouse and pilot will C histopathological examination showing granulomatous lesions with fungi within multinucleated giant cells HE x160 D histopathological examination showing multiple budding yeast form GMS x160 BCD courtesy of the Department of Pathology Faculdade de Medicina de Botucatu UNESP Its observation is easier when stained with lactophenol cotton blue Gomori methanamine silver GMS and periodic acid of Schiff PAS than with hematoxilineosin HE which hardly stain the fungal wall On the other hand HE stain permits the identification of the preserved fungal cells 22 Phylogeny The high prevalence of negative serological tests in mycologically confirmed patients from the Midwest Region using antigens from isolates in the Southwest Region B339 suggested genetic differences among samples from distinct origins 38 In addition genetic differences demonstrated by RAPD were detected in fungal cells isolated from the forearm and face of the same patient at the same admission 39 Then an isolate from a patient of the State of Mato Grosso Midwest Region Brazil was well characterized and due to the differences of P brasiliensis it was coined isolates 550B and 550F Moreover it was demonstrated that all the isolates from Mato Grosso Midwest Region Brazil belonged to the cluster Pb01like 40 The study of these and other isolates led to the description of a new species of Paracoccidioides initially coined Pb01 which received the name of Paracoccidioides lutzii in honour to the researcher who reported the first two cases of PCM 41 In the same study the cryptic species S1 PS2 and PS3 were molecularly characterized 23 Transition from the Mycelial to the Yeast Phase Species of the P brasiliensis complex have the ability to change their morphology from a multicellular filamentous form to a unicellular form when they infect host tissues a process referred to as dimorphism 42 The temperature is the only factor triggering P brasiliensis dimorphism when α13glucan the major cell wall neutral polysaccharide constituent of the pathogenic yeast phase replaces almost entirely the βglucan that comprises the neutral polysaccharide of the vegetative mycelial phase This transition behaves as a mechanism of escape and α13glucan 228 The Open Microbiology Journal 2017 Volume 11 Mendes et al like a virulence factor 3 ECOLOGY 31 Ecological Aspects of P Brasiliensis and P Lutzii Despite important advances in knowledge of the biology of the etiological agents of paracoccidioidomycosis PCM we are far from having the complete picture of relevant biological factors such as the ecology of these agents Few reports are present in the existing literature that address the isolation of these pathogens from the environment 43 47 and while fungal isolation from the faeces of bats and penguins and dog food contaminated with soil has been reported only casual remarks with limited reproducibility have been made 48 51 The lack of outbreaks and prolonged latency period of the disease together with human migration have resulted in the exact infection source remaining unknown 45 An important clue for ecological studies on P brasiliensis was the finding of naturally infected ninebanded armadillo Dasypusn ovemcinctus in endemic areas 13 52 55 The fungus was also isolated from another armadillo species Cabassous centralis reinforcing that armadillos are in constant contact with the pathogen in the environment 56 The systematic recovery of P brasiliensis from armadillo tissues has demonstrated the importance of this animal in PCM endemic areas helping locate hot spots of the fungus occurrence in some environments such as in some restricted andor protected soil conditions in places containing natural and anthropic disturbed vegetation near water sources 53 57 The environment represented by the armadillo burrow and its surroundings associated with biotic and abiotic features may contribute to the development of the fungus saprobic stage in nature as already demonstrated by Terçarioli et al 58 However P lutzii has not previously been isolated from armadillos even when the animals were captured in the central or western regions of Brazil which have been identified as endemic areas for this species 59 On the other hand regardless of whether P lutzii has been isolated from armadillo tissues both P brasiliensis and P lutzii have been molecularly detected in soil and aerosol samples obtained from armadillo burrows and habitats 59 Phylogenetic studies have suggested that P brasiliensis P lutzi and other Onygenalean Ascomycota dimorphic fungi such as Blastomyces dermatitidis Histoplasma capsulatum Coccidioides immitis and C posadasii have evolved in association with animal hosts since ancient times adapting to two distinct ecological niches the first represented by natural saprobic conditions in soil and the second represented by the live tissues of animal hosts 57 60 61 This co evolution with animals may have induced irreversible genetic changes in these pathogens resulting in increased adaptation to biotrophic lifestyles and significant reductions in their saprobic forms Though it is clear that saprobic forms of Pbrasiliensis and Plutzi persist in the environment producing infective propagules conidia that can induce primary infection of the lung via the airborne route it is also quite certain that the saprobic phase may be relatively transitory and occur under only special environmental conditions such as below soil surfaces in burrows and other similarly protected habitats 57 Although there are several indications that P brasiliensis and P lutzii exhibit different geographical distributions this subject is far from resolved While P lutzii isolates have been detected mainly in the central and western regions of Brazil this species has previously been isolated from one patient and detected molecularly in aerosol samples from the southeast region 59 62 63 Experimental studies have suggested that the ability to produce infective conidia differs between genetic groups or cryptic species of Paracoccidioides which in turn may determine the incidence of infection For example the S1 and PS2 genotypes occur sympatrically in Southeast Brazil at a rate of 91 S1PS2 and conidia production has been identified to be higher in S1 than PS2 isolates under laboratory conditions 64 Studies were carried out in PCM patients with the acutesubacute form evaluating the incidence of the cases in relationship with several climatic conections precipitation air temperature absolute and relative humidity soil water storage and Southern Oscillation Index SOI 65 The results suggest that higher water storage two years before exposure may explain the relationship between P brasiliensis and rainfallhumidity Probably there is fungal growth after increase in soil water storage in the long term followed by greater spore release with increase in absolute air humidity in the short term There are indirect evidences that P brasiliensis grows preferentially 220cm below the soil suface a condition that protects from the competitor abundant in the first soil layers Human rural activities remove the level surface of the soil exposing the filamentous spore producing form of the fungus If the absolute humidity is high enough at that moment the conidia and fragments of mycelia are aerosolized It was also described the first welldocumented cluster of cases of the acutesubacute form of PCM with potential relationship with the El Niño Southern Oscillation ENSO phase in 19821983 66 The ENSO behaviour explains the Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 229 rainfall variability not only in the Brazilian equatorial region but also in the Southern The atypically high soil water storage in 19821983 due to precipitations higher than two standards deviations above the mean in the Botucatu region São Paulo state area of this study can be implicated in this finding 4 VETERINARY ASPECTS 41 Infection of Paracoccidioides Brasiliensis in Domestic and Wild Animals Studies of natural infection with P brasiliensis in animals are relatively scarce when compared with studies conducted on human populations Evaluations of fungal infections in animals have been mostly performed using intradermal tests serological surveys histopathological analyses molecular tools and isolation of the pathogen in culture Most of these studies have been carried out in South and Southeast Brazil which are areas of high PCM endemicity Delayed hypersensitivity tests performed using paracoccidioidin as the antigen under study on wild terrestrial coatimundis and Felidae species and arboreal animals weepingcapuchins and marmosets have demonstrated a significantly higher rate of positivity in terrestrial 83 than arboreal animals 22 67 Among domestic animals positivity rates have been reported to be higher in equines 64 than sheep 41 and cattle 40 68 While serological surveys have been performed on different animal species they have predominantly been carried out on dogs In these studies the observed rates of positivity were 78 in dogs from Botucatu São Paulo State Brazil 74 in dogs from São Paulo São Paulo State Brazil 69 and 90 49 and 15 in dogs from rural suburban and urban areas of Londrina Paraná State Brazil respectively 70 Infection of animals by P lutzii was observed in the State of Rio Grande do Sul Brazil 71 The data showed no difference of prevalence between infection by P brasiliensis and by P lutzii Comparison among animals revealed increased prevalence of infection by P brasiliensis and P lutzii in dogs and by both species in wild animals In addition the prevalence of serum positivity varied regarding the geographic origin of the animals Dogs are susceptible to experimental Paracoccidioides infection as was demonstrated by the infection of four puppies 72 Two of the puppies died one week after inoculation and were autopsied Histopathological examination showed granulomas in the lungs spleen and liver and the fungus was recovered in culture The remaining two dogs were evaluated one and five months postinoculation no gross lesions were identified in the organs of these puppies and the fungus was not recoverable Natural disease in dogs was first reported in a female adult Doberman Pinscher from Mogi Guaçu São Paulo State Brazil which exhibited poor general condition and cervical lymph node enlargement 73 The lymph nodes of this dog were biopsied and the histopathological examination revealed active PCM with numerous typical P brasiliensis yeast forms After treatment with ketoconazole total regression of the lymphadenomegaly was observed however clinical recurrence was observed 18 months later and the dog was euthanized but not autopsied The second case was reported in a female adult Doberman Pinscher from Curitiba Paraná State Brazil 74 This dog presented emaciation generalized lymphadenomegaly and hepatosplenomegaly The popliteal lymph node of the dog was biopsied and cultured P brasiliensis was recovered and the animal was successfully treated with itraconazol for two years Molecular tools have facilitated the detection of P brasiliensis DNA in both soil and animal tissue samples 72 73 Using this approach a variety of wild animal species that were roadkilled in PCM endemic areas in São Paulo State Brazil were found to be infected with P brasiliensis including two species of armadillos Dasypus novemcinctus and Dasypus septemcinctus and guinea pig Cavia aperea raccoon Procyon cancrivorus porcupine Sphiggurus spinosus and tayra Eira barbara species 75 The discovery of ninebanded armadillos Dasypus novemcinctus naturally infected by P brasiliensis constituted a landmark finding in the ecological study of this pathogen leading to a better understanding of its possible distribution in nature 13 52 53 Cabassous centralis nakedtailed armadillo has also been found to be naturally infected in Colombia as was demonstrated for the first time by isolating this fungus from armadillo tissues and confirming its identification by sequencing the ITS and gp43 regions 56 Armadillos belong to the order Xenarthra an ancient order of mammals that also comprises sloths and anteaters It is believed that this order has existed in South America for 65 million years and because the order Onygenales originated 150 million years ago it has been proposed that the two orders coevolved in concert 60 Of the 230 The Open Microbiology Journal 2017 Volume 11 Mendes et al xenarthrans armadillos are most important in the ecology of P brasiliensis Paracoccidioidal infections in two species of anteater Tamandua tetradactyla and Myrmecophaga tridactyla were reported for the first time in São Paulo State Brazil and detected by molecular amplification of the ITS region through which the fungus was identified in the lungs liver spleen and mesenteric lymph nodes of each animal 76 The final link in the epidemiological chain connecting P brasiliensis and xenarthran species was formed by identification of naturally acquired PCM disease in a twotoed sloth Choloepus didactylus from Central America The animal was from French Guiana and died during the quarantine period in a pet shop at Monterrey Mexico The diagnosis was confirmed by the presence of granulomatous lesions in the lungs liver spleen and kidneys which exhibited typical multi budding P brasiliensis yeasts 77 These findings show that P brasiliensis and P lutzii infection in animals from areas endemic to the human disease is more common than previously realized thus it is important that veterinarians be aware of the potential for Paracoccidioides infection 5 EPIDEMIOLOGY The occurrence of PCM has been reported from Mexico to Argentina with the largest number of patients being from five countries Brazil Venezuela Colombia Ecuador and Argentina Chile is the single South American country without any reported autochthonous case which is due to climactic conditions that are unfavorable for the fungus survival in the soil In Brazil the Southeast CentreWest and South regions have the largest number of cases PCM affects current or former rural workers who are exposed to intense and continued contact with the soil The disease predominates among males due to the protection conferred by estrogen which inhibits or hinders the transformation of conidia and mycelial fragments into the yeastlike form which is pathogenic 78 80 The epidemiological pressure associated with the predominance of disease among rural male workers can also influence the distribution of cases per gender The malefemale ratio is 1710 among patients with the acutesubacute form AF and 22010 among those with the chronic form CF of the disease PCM predominates among individuals aged 30 to 59 years of age and also among those of mixed race 81 The Infectious Diseases Service of the School of Medicine of Botucatu receives on average 15 new cases of PCM per year 82 Skin tests revealed high rates of infection in several Brazilian areas with no difference as to gender The Botucatu region São Paulo state is hyperendemic and the paracoccidioidal infection can occur at an early age including in 5 yearold children 83 The use of a specific antigen in the skin tests reduces the possibility of cross reactions with other fungal infections The gp43 was used as antigen in a survey performed in individuals from rural settlements in a Brazilian Midwest region demonstrating a prevalence of 458 of infection 82 A study that analysed death certificates detected a greater number of deaths by PCM in the Southeast CentreWest and South regions of Brazil but higher mortality rates in the CentreWest region and the state of Santa Catarina 84 Most deaths correspond to patients over 60 years of age in every region That study also revealed that PCM is the eighth cause of death among the predominantly chronic or relapsing infectious diseases The mortality rate in the state of Paraná is 348 cases1 million inhabitants 85 In the state of São Paulo the mortality rate is 266 158 as primary and108 as an associated cause of death However in the Botucatu area which is in the centrewest region of the state of São Paulo the corresponding rates are 873 489 and 384 confirming its status as hyperendemic area 86 A study on the prevalence of blood types among patients with PCM as a function of the severity of disease and compared to healthy subjects suggested that the red blood cell antigens Jka Jkb Fyb and Leb might play a role in the immunopathology of disease possibly as resistance factors 87 6 GENETIC ASPECTS Infectious diseases are complex traits since acquired and genetic factors related to the host pathogen characteristics and environmental conditions contribute to the outcome risk The finding that only 2 of individuals exposed to species of Paracoccidioides develop PCM 88 favours the hypothesis that a genetic component plays a role in the balance between infection and disease The importance of the host genetics in PCM was demonstrated by experimental models using resistant and susceptible mice The pattern of lesions showed remarkable differences specially related to the extracellular matrix of the granulomatous lesions Resistant mice revealed the coexistence of two types of lesions one type presenting encapsulated nodules constituted mainly of type I collagen and abundant neutrophil burden in areas of massive fungal destruction the other type showed residual characteristics with sparse collagen deposits containing xantomatouslike macrophages with degenerated fungi On the other hand susceptible mice showed only one type of lesion with less tendency to encapsulation presence of reticular type III collagen many plasmocytes and large number of budding yeasts with no evidence of fungal destruction 89 In addition in the same experimental model the early influx of neutrophils to the lungs was higher in susceptible rather than in resistant mice infected with P brasiliensis while neutrophil depletion resulted in decreased survival time of susceptible but not resistant mice showing the influence of genetic factors on the immunoprotective and immunoregulatory role of these cells in PCM 90 Data on human genetic susceptibility for PCM are scarce and no result was replicated or validated Some candidate genes have been already tested but the short number of individuals evaluated has been a limitation for these studies In addition the selection of controls for studies on genetic association is a critical point and the exposure to the pathogen should be taken into consideration Thus the ideal controls for PCM studies are individuals living and working in rural areas of risk for a long time Positive intradermal test performed in healthy individuals with specific antigens should be used as criteria for control selection since this test has been a good marker to confirm a previous fungal exposure 91 Fig 2 Phylogeny and recombination in Paracoccidioides Two methods were used to examine strain relationships originating from across South America A using 614570 SNPs including a phylogenetic network constructed with SplitsTree4 B and a Bayesian calibrated phylogeny constructed with BEAST C bootstrap values from maximum likelihood phylogeny constructed with RAxML were included for major subdivisions Both methods show evidence of five distinct lineages in P brasiliensis S1 blue which is divided into two groups S1a dark blue and S1b light blue PS2 green PS3 red and the recently described PS4 purple Also this phylogeny supports the divergence between P brasiliensis and P lutzii Pl orange as a different species In addition the phylogenetic network of P brasiliensis suggests patterns of recombination red branches Note from Muñoz JF Farrer RA Desardins CA et al Genome diversity recombination and virulence across the major lineages of Paracoccidioides 2016 mSphere 15 e0021316 232 The Open Microbiology Journal 2017 Volume 11 Mendes et al IL12IL23IFNγ axis also suggests a role of the host genetics in PCM 92 These disorders are genetic defects which predispose to severe forms of some infections mainly caused by nonpathogenic mycobacteria known as Mendelian susceptibility for mycobacterial diseases MSMD Thus genes related to this pathway are candidates to association with susceptibility for PCM Variants at IFNG and IL12B genes were not associated with clinical forms of PCM in Brazilian patients while IL12RB1 presented one polymorphism associated with the disseminated chronic form 93 However more variants at these genes should be tested in order to elucidate its participation in determining the clinical form Polymorphisms at IL4 gene were associated with susceptibility for PCM and with the production of the cytokine by PBMC after specific stimulus 94 95 A polymorphism located at the promoter region of IL10 was also associated with PCM in a specific Brazilian population 96 Variants at CTLA4 TNF and IFNG genes were not associated with PCM 94 96 97 but class I and class II HLA alleles were associated with PCM The first studies demonstrated a higher frequency of HLAA9 e HLAB13 alleles in Colombian patients 98 In Brazilians B40 Cw1 A2 B7 and B21 alleles had higher frequencies in PCM patients 99 100 In another Brazilian study the DRB111 allele was associated with the unifocal chronic form of the disease 101 These data should be carefully evaluated because of the ethnic differences of these patients as to the region they are from Since the clinical forms of PCM are dependent on the host adaptive immune response the genes driving such response are strong candidates to determine a specific clinical form Thus data from patients with the AF and the CF should be analyzed separately Largescale genetic studies in PCM are required to elucidate the architecture of the disease aiming better strategies of control diagnosis and treatment Smoking which is highly common among patients with PCM increases the risk of pulmonary PCM 10fold and reduces by 8 years the patients age at the onset of symptoms 87 An evaluation using multilocus sequencing studies maximum likelihood and Bayesan analyses carried out on isolates within the Paracoccidioides genus confirmed the existence of two distinct species P brasiliensis and P lutzii and supported that P brasiliensis isolates are clustered into five distinct lineages S1a S1b PS2 PS3 and PS4 The S1b lineage includes the reference strain Pb18 and the S1a lineage is split into two subclades PS3 includes isolates from Colombia and Pb 339 an isolate from southeast Brazil used in the preparation of antigen for diagnostic tests The Fig 2 shows the phylogeny and recombination in Paracoccidioides and the regional distribution of the isolates in South America The clinical impact of these findings are related to the preparation of antigens for serological diagnosis and followup because the sensitivity of these tests is higher when a specific preparation is used In addition clinical manifestations radiological findings and response to treatment should be evaluated regarding to the P brasiliensis lineage 102 7 PATHOGENESIS 71 Natural History The lungs are the usual portal of entry for Paracoccidioides sp into the human body spores reach the terminal bronchioles and alveoli causing areas of pneumonitis From these areas the fungus spreads by the lymphatics to the paratracheal and parabronchial lymph nodes where it triggers a granulomatous reaction Fig 3 The areas with pneumonitis constitute the parenchymal pole of the paracoccidioidal infection and the affected regional lymph nodes constitute the lymphatic pole Together the parenchymal pole ascending lymphangitis and satellite lymph node affection is known as PCM primary complex 8 103 The hosts immune response to infection with Paracoccidioides sp determines the progression of the hostparasite interaction When the immune response is satisfactory the body blocks infection at the level of the primary complex and its eventual metastases In such cases the inflammatory reaction recedes and scars are formed which may be sterile or contain viable albeit latent fungi These patients exhibit infection only which is detectable through a positive intradermal reaction to paracoccidioidin Depending on the balance between the host parasite and environment the fungi may remain latent for many years occasionally for life However after a variable usually long period of time any imbalance between these factors may result in reactivation of latent foci a phenomenon known as endogenous reinfection which triggers disease Because a large portion of patients remain in continuous contact with the soil after the initial exposure to the fungus it is difficult to assess the contribution of a new infection ie the socalled exogenous reinfection to the triggering of disease In turn the development of disease in patients many years after having moved away from an endemic area confirms the relevance of endogenous reinfection Fig 3 Natural history of paracoccidioidomycosis Note from Franco M Mendes RP MoscardiBacchi M RezkallahIwasso M Montenegro MR Paracoccidioidomycosis Baillières Clin Trop Med Commun Dis 1989 41 185 220 When the immune response is insufficient at the time of primary complex formation the fungi multiply and spread first by the lymphatic system and then the haematogenous route to various organs and systems In such cases the disease manifests immediately following infection ie after the first contact with the fungus PCM disease might progress into death or cure Cure is associated with scar formation in the affected organs which may eventually cause sequelae among which lung fibrosis and emphysema with consequent functional impairment stands out The scars may be sterile or contain viable fungi which might cause relapse Although rare cutaneous inoculation can also be a portal of entry for Paracoccidioides sp However to establish that a skin lesion was caused by direct inoculation the occurrence of local trauma in the affected area two to three weeks before the appearance of regional lymphadenopathy should be confirmed and lung compromise ruled out In addition to these criteria other findings are also relevant including a good overall state of health the absence of other clinical manifestations attributable to PCM the presence of compact granulomas on histopathological examination of lesions and a strongly positive intradermal reaction 72 Virulence and Escape Mechanisms The yeast phase is the pathogenic form of the P brasiliensis High concentrations of α13glucan and low concentrations of galactomannan in the cell wall of the yeastlike forms are correlated with P brasiliensis virulence 234 The Open Microbiology Journal 2017 Volume 11 Mendes et al 104 105 In a murine model gp43 antigenaemia led to depression of the cellmediated immune response 106 In addition gp43 antigenaemia was adequately demonstrated in patients and may last a very long time up to two years in patients with the acutesubacute form of disease 107 These findings suggest that gp43 might have a considerable immunomodulating effect maintaining the depression of the cellmediated immune response and high serum antibody levels The immunogenicity and pathogenicity of P brasiliensis samples freshly isolated from patients with PCM were assessed and compared to the severity of disease 108 The results showed a direct correlation between severity of disease and virulence especially in the patients at both extremities of the severity spectrum mild and severe By contrast the immune response was found to be a host characteristic with the infecting fungi playing a secondary role 73 Interaction of Paracoccidioides Brasiliensis Complex with Host Cells The respiratory epithelium represents the primary site at which contact between fungus propagules and hosts occurs The cells involved in human Paracoccidioides sp infection include alveolar macrophages and alveolar epithelial cells 109 Although epithelial cells serve as a relatively passive physical barrier to infection they may contribute more actively to the signalling events that occur during immune responses 109 Furthermore epithelial and endothelial cells may serve as a reservoir for the fungus protecting them from macrophages and other immune system cells 110 The migration of pathogenic yeasts to endothelial cells is considered to be a prerequisite for multiple organ invasion and fungus dissemination 109 111 The use of mammalian cell culture techniques has provided unique insights into these hostfungus interactions A cell line derived from human alveolar epithelial cells A549 cells has been used as an in vitro type II pulmonary epithelial cell model as have been Vero and HeLa cells 112 These models have been developed to study the processes that occur between initial hostParacoccidioides sp contact and the events that culminate when fungal cells enter the host 113 Fungal adhesion processes have been reported to vary between strains and correlate with virulence 114 and strains that are more virulent in animals have been found to exhibit enhanced adhesion in vitro 115 The ability of pathogens to colonize their hosts is highly dependent upon the mechanisms that allow the pathogen to overcome the physical and immunological barriers imposed by the host To avoid rapid clearance pathogens may quickly and effectively adhere to host cells The capacity of the type of the cells to interact with each other in an orderly manner depends on multiple adhesive interactions between cells and their adjacent extracellular environment mediated by cell adhesion molecules 116 117 that function as cell surface receptors that can trigger physical and biochemical signals that regulate a great numbers of functions such as cell proliferation gene expression differentiation apoptosis and cell migration and are used as a gateway to some pathogens 118 121 The internalization of many pathogenic microorganisms by epithelial cells may be associated to the ability of these organisms to induce this process forcing the activation of phagocytosis mechanisms and resulting in these cells behaving like unprofessional phagocyte thereby providing a mechanism by which yeast cells can evade the professionalphagocytes and potentially facilitating the dissemination ofpathogens 122 For this process to occur specific extracellular signals stimulate cytoskeleton rearrangement at the site in which contact with the microorganism occurs 123 124 involving integrins and the cytoskeleton 113 125 The involvement of integrins in these processes could be linked to the interaction between fungal cells and the host lipid membrane rafts and associated with the production of certain types of interleukins Therefore the disruption of epithelial cell membrane rafts by nystatin for example has been found to be associated with decreased IL6 and IL8 levels in Paracoccidioides spA549 cell cultures Therefore these interactions have been found to be associated with increases in the hosts α3 and α5 integrins levels and the clustering of receptors onto membrane rafts suggesting that Paracoccidioides sp may modulate host inflammation 126 The structures of the cytoskeletons of pulmonary epithelial cells and keratinocytes and their morphological features including actin tubulin and cytokeratin could be affected by the interactions between the host and Paracoccidioides sp 127 128 Keratinocyte parasitism may represent a possible mechanism by which fungal cells can evade local immune mechanisms 129 Furthermore cytochalasin D and colchicine treatment have been found to reduce Paracoccidioides sp invasion indicating the functional involvement of microfilaments and microtubules in this process 127 130 Some Paracoccidioides sp proteins referred to as adhesins may be mediating the cell invasion process The 43 kDa glycoprotein gp43 may also participate in cytokeratin degradation leading the loss of the filamentous characteristics Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 235 and facilitating the invasion of the host 130 134 Additionally the 1433 adhesin has been recognized to have the capacity to cause structural modifications in host cells thereby influencing polymerization of the cytokeratin microfilaments of actin 135 139 Members of the Rho GTPase family of proteins have been observed to regulate the dynamic organization of the cytoskeleton and membrane traffic related to physiological processes such as cell proliferation motility polarity and growth 140 thus these proteins may play an important role in the interaction between fungal and mammalian cells The activation of the tyrosine kinase PTK receptors that stimulate Rho GTPase subsequently activates the Ras pathways and MAPKs 140 during a previous evaluation of this process pretreatment of epithelial cells with genistein resulted in a significant inhibition of fungal invasion suggesting that the inhibition of PTK is important in signal transduction during early events in the epithelial cell adhesion and invasion processes of Paracoccidioides sp 141 Another important family of kinases involved in the Paracoccidioideshost interaction is the Src family SFKs and the hostfungus interaction process may involve the activation these kinases and extracellular signalregulated kinase 12 ERK12 in the affected epithelial cells These data indicate that epithelial cell membrane rafts are essential for the adhesion to and activation of cell signalling molecules by Paracoccidioides sp 142 for example cytokine secretion was reported to be dependent upon p38 mitogenactivated protein kinase MAPK cJun NH2terminal kinase JNK and extracellular signalregulated kinase ERK 12 activation and the secretion of IL8 and IL6 that is promoted by this fungus have been reported to be dependent upon activation of p38 MAPK and ERK 12 in A549 cells 143 The ability of these pathogens to induce apoptosis may be an important virulence factor since it reduces the hosts defence mechanisms 144 Paracoccidioides sp induce apoptosis when they invade epithelial cells or phagocytes benefiting the intracellular survival of this fungal species 127 145 146 Furthermore the induction of macrophage apoptosis has been found to be associated with expression of caspase2 3 and 8 147 Moreover Paracoccidioides sp may modulate epithelial cell A549 apoptosis via the expression of apoptotic molecules such as Bcl2 Bak and caspase3 supporting the hypothesis that apoptosis may be induced by the fungus to promote its survival and dissemination 148 149 More recently Silva and colleagues 150 showed that the 1433 and gp43 adhesins had substantial influences on this process Additionally apoptosis may be mediated by FasFasL and CTLA4 was identified to be involved in modulating immune responses in patients infected with PCM 151 To further increase the understanding of Paracoccidioideshost interactions the use of mammalian cells models may facilitate the establishment of increased knowledge related to how fungushost interactions have evolved to enable the fungi to evade the immune system of the host and why Paracoccidioides sp are the organisms associated with the highest incidence of mycosis in Latin America 130 74 Pathology Histomorphologic View The pathological findings of PCM will be focused on the pulmonary involvement It is characterized by granulomatous inflammatory processes during which the intensity and morphologic pattern depend on the immune status of the host duration of infection without treatment and virulence of the fungus 152 Typically performing hematoxylin and eosin staining on pathological specimens from immunecompetent patients reveals multiple wellformed granulomas composed of cohesive clusters of histiocytes and organized centrally with peripheral admixed lymphocytes however some eosinophils andor neutrophils have been detected Fig 1C 153 154 These histiocytes are also characterized by broad foaming cytoplasm called epithelioid and giant cells are occasionally observed in their inflammatory infiltrate 153 Both cells may engulf a relatively small number of P brasiliensis forms in granulomas however they may also course freely within the connective tissue Moreover necrosis is not frequently observed but may occur in some cases According to the grade of immunity granulomas may be extensive with dispersed macrophages lymphocytes and plasmocytes On the other hand immunocompromised patients may develop pneumonic reactions with diffuse alveolitis composed predominantely of neutrophils and affecting both the hilar and peripheral regions of the lungs 153 155 Potential P brasiliensis infections are generally evaluated based on typical histomorphological features through the use of light microscopy in appropriate clinical settings A multinucleated cytoplasm that is separated from the cell wall with a clear space or halo may sometimes be recognized in HEstained sections To better diagnosis PCM Gomoris methenamine silver staining GMS should be performed to determine the morphological aspects of this fungus 236 The Open Microbiology Journal 2017 Volume 11 Mendes et al Through the use of GMS and other staining methods such as Papanicolaou and periodic acidSchiff PAS staining the yeast cells of P brasiliensis which are spherical vary markedly in size and have multiple buds or a pilot wheel appearance may be revealed 155 In general the injuries resulting from PCM are centred on the bronchovascular axes of the proximal airways and associated with radiological findings consistent with a butterfly wing pattern In patients with chronic disease the granulomatous inflammatory process promotes myofibroblastic activation and increased extracellular matrix deposition 155 Consequently parenchyma and vascular lung remodelling result in improvements in radial fibrosis traction bronchiectasis and lung architectural distortion The final outcome is a pulmonary fibrosis sometimes accompanied by cor pulmonale and death 152 8 AUTOPSY FINDINGS Table 1 describes the prevalence of lesions in several organs as found in necropsy studies The data indicate the predominant involvement of the lungs lymph nodes mucous membrane of the UADT and adrenal glands 156 163 Table 1 Prevalence percentage of organs involved in patients with paracoccidioidomycosis Study of autopsies BenaimPinto et al 1961 N50 Del Negro 1961 N56 Brass et al 1969 N36 Dillon 1972 N14 Salfeder 1969 N11 Defaveri et al 2002 N13 N40 Organs Acute Chronic Lungs 696 678 750 420 1000 1000 975 Limph nodes 677 643 330 280 727 500 Oral mucosa pharynx larynx 556 411 400 182 700 Adrenals 567 482 800 570 363 750 630 Central nervous system 22 125 210 Liver 290 375 270 210 455 1000 Spleen 176 393 27 210 545 Skin 313 393 27 640 Kidneys 62 196 83 140 91 Bowels 234 284 27 Bone marrow 750 Heart 20 27 70 91 BenaimPinto H Mycopathologia 1961 15 90 114 Del Negro G tese Faculdade de Medicina da Universidade de São Paulo São Paulo SP 1961 Brass K Mycopathologia et Mycologia Applicata 1969 37 119 138 Dillon NL tese Faculdade de Medicina da Universidade de São Paulo São Paulo SP 1972 Salfelder K Doehnert G Doehnert HR Virchows Arch Abt A Path Anat 1969 348 51 76 Defaveri J Joaquim A Ann Rev Biomed Sci 2002 special issue 111 9 IMMUNE RESPONSE The onset progression and clinical outcomes of PCM are influenced by environmental factors the hosts immune responses and genetic background 164 Compelling evidence suggests that immunity against Paracoccidioides sp is based on three major principles 1 PCM is an endemic disease and affects healthy individuals ie those without immunosuppressive underlying conditions such as neoplasia or use of immunosuppressive drugs 2 the adaptive immune response against Paracoccidioidesspecific antigens is deficient and may modulate the immune responses to other antigens 165 and 3 host responses are dependent upon gender nutritional status size of inhaled inoculum and possibly genetic background In Paracoccidioides sp cell death may be induced by hydrogen peroxide H2O2 produced by macrophages 166 which is enhanced by the T helper 1 Th1polarized immune response 167 Effector Th1 lymphocytes are recruited to the site of infection and release IFNy which enhances macrophage activation Therefore disturbances in the orchestration of these mechanisms may lead to the onset and progression of disease The duration of the symptomatology of PCM is short in patients with the AF ranging from a few weeks to some months median of 2 months 168 and prolonged in patients with the CF usually higher than 6 months 169 Independent of the clinical form of PCM hosts initiate an adaptive immune response and by the time of patient admission this response has already become polarized Although the initial events of the Paracoccidioideshost interaction have been well explored using experimental models in the present review we mainly focused on the immune features observed in PCM patients PCM patients show a wide spectrum of clinical manifestations which are correlated with the type of immune response activated 170 171 The adaptive immune response involves highly specific interactions between immune cells and soluble factors such as antibodies cytokines and fungal antigens In general the clinical forms of PCM exhibit dichotomous Th responses whereas patients with the AF of the disease have abundant antibodies but poor to nil T cellcell mediated immune responses patients with the CF of the disease demonstrate good T cell responses as indicated by skin tests and in vitro correlates of T cell immunity 170 171 An overview of the polarization of adaptive immune responses in PCM patients is shown in Fig 4 Fig 4 Overview of the polarization of adaptive immune in PCMA Activation of T helper Th subsets Paracoccidioides antigenprimed dendritic cells DCs migrate to the lymph node where they present processed antigens to naïve Tn cells that differentiate into one of Th lymphocyte subsets Th1 Th2 Th9 and Th17 depending primarily on cytokines present in the extracellular environment For Th1 polarization IL12 from DCs and macrophages and IFNγ from NK cells activate STAT1STAT4 signaling to induce expression of the Th1specific transcription factor Tbet For Th17 polarization IL6 IL1β TGFβ and IL23 are required to induce expression of the Th17specific transcription factor RORβτ through STAT3 signaling For Th2 polarization IL4 from DCs activates STAT6 signaling to induce expression of the Th2specific transcription factor GATA3 For Th9 polarization IL4 and TGFβ are required to induce expression of the Th9specific transcription factor PU1 B Effector phases of T cell responses Th1 Th17 Th2 and Th9 clones could be distinguished mainly by the cytokines produced by the cells Th1 cells release high amounts of IFNγ and TNFα that classically activate macrophages M1 resulting in fungal elimination Th17 cells secrete IL17 and IL22 that recruit neutrophils and monocytes Neutrophils cells act by generate reactive oxygen species ROS release of neutrophil extracellular traps NETs that result in fungal elimination Monocytes have been studied in PCM by induce high levels of inflammatory cytokines such as TNFα and IL1β and growth factors such as TGFβ and fibroblast growth factor FGF Th2 show several functions that depend of each secreted cytokine IL4 induces activation of B cells and subsequent production of immunoglobulins IL5 triggers recruitment of eosinophils and IL13 is involved in the deactivation of macrophages termed alternatively activated macrophages M2 that results in fungal growth and also in tissue repair Th9 release IL9 and IL21 that act in synergy with Th2 to produce antibodies The Th2 Th9 type of immune response is characteristic of the acute clinical form of PCM Table 2 Patients with the AF of the disease exhibit high IL4 IL5 and IL9 levels and nonreactive paracoccidioidin skin tests This finding reflects the marked depression of cellularmediated immunity that occurs in these patients Furthermore these patients produce large amounts of antigenspecific IgA IgE and IgG4 172 an antibody isotype that has been found to exhibit diminished complement fixation capacity and a low affinity for the FcR receptors present in phagocytes resulting in poor phagocytes is by macrophages and subsequent fungal multiplication and dissemination In patients with the CF of the disease following disruption of the prolonged fungushost equilibrium the reactivation of latent foci endogenous reinfection leads to disease progression Regardless the Th1 response is more preserved in these patients Table 2 since the majority of them has reactive paracoccidioidin skin tests except for those with severe forms of the disease In addition these patients exhibit increased production of proinflammatory cytokines Fig 5 Proposed overview of fibrogenesis in PCM The uncontrolled deposition of collagen fibers types I and III during a reparative andor reactive process is recognized as fibrosis and the fibroblasts are the main cell involved in the production of collagens upon stimulation During prolonged chronic inflammation macrophages are classically activated M1 by Th1 cells proinflammatory cytokines andorimmunocomplexes These cells show intense production of cytotoxic metabolites to kill pathogens release proinflammatory cytokines induce tissue necrosis and recruit phagocytes Fresh monocytes from peripheral blood and monocytederived macrophages release inflammatory cytokines and growth factor that result inmacrophage activationsand fibroblast differentiation Simultaneously due to the constant tissue damage others macrophages are alternatively activated M2 to stimulatetissue repair These cells promote the elimination of cellular debris activation of fibroblast and downregulate the activities of metalloproteinases enzymes implicated in extracellular matrixcollagen degradation promoting deposition of collagen fibers On the other hand M1polarized macrophages upregulate metalloproteinase activities resulting in less collagen deposition Besides the nonregulated constant and prolonged reparative and reactive processes involved in fibrogenesis do not rule out the action of molecules of Paracoccidioides in the activation of fibroblast Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 239 CD14 CD16 inflammatory monocytes 178 The immunological alterations observed in patients with the CF of the disease during and after treatment may be associated with hypoxia due to pulmonary fibrosis and emphysema Activation of some transcription factors such as hypoxiainducible factors HIF 179 may induce growth factor signaling proinflammatory cytokine release costimulatory molecule expression and lymphocyte proliferation 180 181 Typically patients with the CF of the disease present with fibrosis at their initial admission 182 and show enhanced production of TGFβ1 and the basic fibroblast growth factor FGFb as shown in Table 2 Previous autopsy findings for PCM patients have demonstrated that pulmonary fibrosis is characterized by extensive areas of collagen deposition in the hilar region and the involvement of other structures such as the lymph nodes bronchi and arteries These collagen fibersfrequently border granulomas and extend to the bronchi and nearby blood vessels The proliferation of reticular fibers collagen III in the alveolar septa also occurs including fibrotic areas of the granulomas 153 Though many challenges remain to be overcome regarding PCM fibrogenesis accumulating evidence suggests that fibrosis occurs as a result of the prolonged inflammation constant antigen stimulation and persistent parenchymal injury that induce the natural wound healing process 183 Chronic inflammation may also induce tissue damage and parenchymal cell death due to necrosis or apoptosis and local production of cytokines and chemokines activate neighbouring cells to produce pro inflammatory cytokines and profibrogenic growth factors such as TGFβ1 and FGF b These mediators stimulate the production of collagen by fibroblasts establishing fibrosis An overview of PCM fibrogenesis is proposed in Fig 5 Table 2 Immunological profiles among paracoccidioidomycosis patients with acutesubacute FA and chronic FC forms and healthy individuals controls according to the production of cytokines by different subpopulations of leukocytes in response to P brasiliensis antigen PbAg Leucocyte Subsets Profile Cytokines No Stimulus PbAg References Control FA FC Control FA FC PBMC Th1 IFNγ IL2 IL12 171 184 185 Th2Th9 IL4 IL5 IL9 171 185 186 Treg TGFβ1 IL10 171 184 186 Th17Th22 IL17 IL22 0 0 NR NR NR 171 Proinflammatory TNFα IL6 NR NR NR 186 CD4 Th1 IFNγ IL2 TNFα NR NR NR 187 CD8 Th1 IFNγ IL2 TNFα NR NR NR 187 Monocytes Proinflammatory TNFα IL6 IL1β IL12 MIP1α H22 NR 178 187 189 Antiinflammatory IL10 TGFβ1 NR 178 187 189 Profibrotic FGFb TGFβ1 NR NR 178 189 Alveolar macrophages Proinflammatory H22 NR NR NR NR 188 PBMC peripheral blood mononuclear cells NR not realized 0 absent mild moderate intense 240 The Open Microbiology Journal 2017 Volume 11 Mendes et al 10 CLINICAL MANIFESTATIONS As a systemic mycosis with remarkable tendency to spread and affect any organ or system PCM exhibits polymorphous clinical manifestations For this reason PCM infection is often confounded with other diseases especially among females and younger patients In general patient complaints include feeling unwell anorexia and weight loss which might be so severe as to cause cachexia Fever is occasionally present and should be considered a sign of greater severity The clinical manifestations associated with the involvement of various organs are described below followed by the classification of the clinical forms of disease 101 Involvement of Organs and Systems 1011 Lungs Lung involvement is particularly relevant due to its high frequency and occurrence of residual fibrosis as well as because the lungs are the portal of entry for P brasiliensis in almost all of the patients The first case of pulmonary involvement by PCM was reported in 1911 190 and the first case with exclusively affected lungs without clinical manifestations compatible with extrapulmonary lesions was published eight years later 191 The relevance of lung participation was recognised only in 1946 when it was detected in 84 of 25 autopsied cases 15 An assessment of patients with PCM who were nonsmokers and did not exhibit any other respiratory disease reported cough in only 57 of the cases and expectoration in 50 The sputum was almost always mucous but bloody in some cases 11 In general the patients did not report chest pain 192 Dyspnoea the most frequent complaint first appeared on heavy exertion and had a progressive character manifesting even at rest Lung involvement however can also be asymptomatic Physical examination of the lungs does not yield many signs even among patients with severe respiratory symptoms characterising a clinicosemiological dissociation The respiratory examination may be normal in up to 43 of patients with PCM lung lesions 192 Plain chest radiographs primarily indicate interstitial or mixed alveolarinterstitial lesions with a predominance of interstitial abnormalities These lesions are usually bilateral parahilar and symmetrical most often located on the middle third of the lungs The upper third is affected in approximately onethird of cases and the apex in half bilaterally Among the interstitial lesions the reticulonodular ones predominate 193 Alveolar or mixed lesions with predominance of the former are also bilateral parahilar and symmetrical usually sparing the lung apex and base The overall aspect is evocative of butterfly wings an image highly suggestive of Paracoccidioides infection although its prevalence is low Fig 6 In addition to the aforementioned patterns the radiological abnormalities might bear resemblance to a tumour pneumonia or a cavitated mass 154 193 Occasionally the radiological findings mimic those observed in tuberculosis Lung cavities were first described by Fialho 15 being characterised as irregular excavations of up to 20cm in diameter and containing a viscous exudate The pressure exerted by neighbouring tissues narrows the cavities down to tortuous slits In association with severe parenchymal involvement this morphology makes their visualisation on plain chest radiographs difficult However these abnormalities are well identified on conventional chest tomography planigraphy where they appear as multiple rounded lesions usually being smaller than 20cm at the largest diameter and exhibiting thick walls Some of the cavitated lesions may be confluent 194 Involvement of the hilar and mediastinal lymph nodes was also detected on autopsy 15 findings which are seldom detected on plain chest radiographs The severe compromising of the parenchyma which is most evident close to the hila impairs the observation of hilar structures However in 50 of the cases chest planigraphy is able to reveal lymph node enlargement 194 Computerised axial tomography of the chest represented a major contribution to the understanding of PCM lung lesions In untreated patients nodules predominate especially small nodules Other findings include septum thickening thick lines alveolar opacities blocks of fibrosis bronchial wall thickening bronchiectasis and cavities without fluid Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 241 content 195 Shortly after the onset of treatment the frequency of bronchiectasis bullae and diffuse emphysema tends to increase 195 On highresolution computerized tomography HRCT thickening of the interlobular septa is the most frequent finding 92 of cases but is sparse and low intensity Thickening is followed in frequency by aareas of emphysema 69 b areas of groundglass attenuation 62 c bronchial wall thickening 54 d tracheal dilation 46 e nodules 39 cavities architectural distortion spiculated pleural thickening and parenchymatous bands 31 and f areas of consolidation intralobular reticular thickening and axial interstitium thickening with bronchovascular distortion 23 196 A clinicoradiological dissociation characterized by a low frequency of respiratory complaints among patients with pulmonary involvement sometimes extensive on radiological examination has been a frequent finding 8 12 16 103 Fig 6 Chest radiograph of a patient with the chronic form of paracoccidioidomycosis in anteropsterior and laterak view showing bilateral and symmetrical alveolar lesions before A and C and after B and D treatment Pulmonary involvement is rare among young patients however it may occur in 5 to 11 of cases 26 197 199 For this reason diagnosis is confirmed only on autopsy Thus in endemic areas this possibility should be taken into consideration whenever a patient exhibits epidemiological antecedents for PCM or the clinical progression is not satisfactory after the use of antimicrobial therapy for common lung disorders Pulmonary function is usually abnormal with the obstructive pattern being the most frequent followed by a mixed pattern very few patients exhibit a restrictive pattern 200 Hypoxaemia occurs in almost all patients and the alveolar arterial oxygen gradient is increased in practically all cases reflecting a predominance of perfusion over ventilation Some data suggest that the air and blood distribution as well as diffusion might be altered in the very early stage of the disease Patients who exhibit an obstructive pattern present with early airway involvement as well as changes in the ventilationperfusion ratio alveolar diffusion and ventilation These abnormalities were also detected in patients with a mixed pattern showing that obstructive lung disorders predominate in PCM The findings on spirometry suggest that 242 The Open Microbiology Journal 2017 Volume 11 Mendes et al bronchial involvement predominates in PCM especially at the level of the bronchioles or of the peribronchiolar connective tissue both in the early and late stage of disease these changes have no relationship to smoking 191 These suggestions are based on a careful autopsy study that evidenced granulomas and areas of fibrosis surrounding the bronchi which were attached to other bronchi and blood vessels via fibrous septa 160 The regression of radiological lesions after the onset of treatment is not attended by recovery of pulmonary function 201 As the proliferation of collagen and reticulin fibers is not always associated with the occurrence of granulomatous reaction but with the presence of P brasiliensis it is suggested that the fungus per se triggers reticulin proliferation 160 The initial respiratory symptoms decrease or fully disappear after the onset of treatment In general patients complain of persistent morning cough attended or not by hyaline expectoration Many patients initially exhibit dyspnoea on heavy exertion which might become worse appearing with moderate or even mild exertion Plain chest radiographs reveal lung sequelae characterised by fibrosis diffuse or bullous emphysema and occasionally pulmonary hypertension Chest computerized tomography shows alveolar opacities 24 of cases nodules 38 mainly small septal thickening 100 bronchial wall thickening 89 usually mild bronchiectasis 41 usually mild bullae 59 diffuse emphysema 70 and pleural thickness 65 Evidence of cavities and honeycomb lesions are infrequent Patients usually do not exhibit hilar or mediastinal lymph node enlargement 182 The pulmonary function is seldom normal 85 of patients exhibit the obstructive pattern and the frequencies of mild moderate and severe degrees of obstruction are equal Hypoxaemia occurs in approximately onethird of cases as a sequel 182 10111 Pleura Pleural involvement is detected in only 2 of cases on plain chest radiographs and is characterised by small effusions and thickening 193 202 Pleural involvement is quite rare as pleural effusion 202 or spontaneous pneumothorax 203 Pleural effusion in PCM was observed predominantly in patients with the chronic form most of them presenting no comorbidity 202 The pleural effusion can be caused by alterations of the permeability due to its paracoccidioidal involvement arisen from a parenchymal process Another possibility is the inflammatory injury to the visceral pleura microcirculation that disrupts the pleurolymphatic drainage It is worth noting the finding that 60 of the autopsied patients presented thickening of pleura without an effusion 15 The diagnosis of the paracoccidioidal pleural involvement depends on the identification of the aetiological agent in tissue fragments andor pleural fluid As cigarette smoking is a risk factor for PCM and CPOD and CPOD can cause by itself secondary spontaneous pneumothorax it is difficult to attribute this pleural disease only to PCM However the odds of pneumothorax in PCM patients is 43 times higher than in cases of CPOD 203 The rupture of the subpleural emphysematous bullae or necrotic cavities caused by a sudden increase in airway pressure can be caused by caugh allowing for communication between the pulmonary airways and the pleural space The spontaneous pneumothorax has been observed in patients with active nontreated PCM in cases of relapse and in patients with apparent cure 203 Sudden onset of dyspnoea chest pain and decreased or absent breath sounds on lung auscultation are the most important clinical manifestations of pneumothorax 10112 Dermatological Aspects Correlations Between Clinical and Histopathological Findings in Paracoccidioidomycosis In patients with systemic mycosis such as PCM the presence of cutaneous and mucosal lesions are initiators of specific diagnoses Taken together the characteristics of these lesions including their number clinical morphology and accessibility for biopsy and collection of material for direct examination and culture provide valuable indicators that may enable early diagnosis In a case series including 152 consecutively enrolled patients with PCM who were followedup at a dermatologic university service the presence of specific cutaneous lesions was observed in 612 of patients and the presence of specific mucosal lesions was observed in 585 of patients 204 Overall 908 of patients included in the afore mentioned study presented with cutaneous andor mucosal lesions accompanied by lung or other systemic involvement These numbers highlight the importance of these lesions as valuable indicators for the establishment of diagnoses Cutaneous lesions generally occur as a consequence of haematogenous fungal dissemination usually originating from the lungs or contiguously evolving from mucosal lesions and often presenting with lip involvement Remarkably Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 243 these lesions arise from direct inoculation of the dermal tissues with Paracoccidioides sp The most common topographic locations of cutaneous lesions are the head and neck 476 inferior limbs 218 trunk and superior limbs 149 and genitals 07 204 Cutaneous lesions most frequently occur in patients with the chronic adult form of the disease but may be identified in patients with the acutesubacute juvenile form of the disease Mucosal lesions are mainly observed on the buccal mucosa and among patient with chronic PCM Lesions on the buccal mucosa are most commonly identified on the gingiva soft palate lip and jugal mucosa 204 205 Lesions occurring on the tongue or tonsils are less commonly observed The clinical features of cutaneous lesions occur as a result of tissue responses to the presence of fungal cells in the dermal tissues This hostparasite interaction is a dynamic process and modification of the immune response may result in the formation of different types of clinical lesions over time in the same patient or differences in the number and clinical features of lesions among diverse patients Ulcers are the most prevalent type of lesion and may arise from pre existing solid lesions such as papular nodular or verrucous lesions or as a consequence of inflammatory events that occur in response to the presence of the fungal cells in the dermal tissues Histologically PCM is characterized by granulomatous inflammatory responses that occur around one or more fungal cells Pedagogically the existence of two polarized clinical expressions of the disease one hyperergic and the other anergic could be conceived a concept first proposed by Lacaz in 1982 and more thoroughly explored by Del Negro et al in 1994 206 Between these polarized forms many possible clinical expressions may occur based on hostparasite interactions On the hyperergic side of the spectrum we would expect patients to have fewer lesions and possibly sarcoid reactions with granulomas that are compact and made up of giant and epithelioid cells In these patients the INFγ and IL12 Th1 immune responses 207 and cells expressing the Foxp3 and CD25 markers may predominate 208 In these cases the inflammatory response is rich in lymphocytes which surround the epithelioid cells and poor in polymorphonuclear leukocytes and the fungal cells are few and contained by inflammatory responses Figs 7A B When affected by the anergic form of the disease patients often present with poor clinical and nutritional status and a large number of cutaneous lesions that are initially acne form but then evolve into ulcerative and necrotic lesions In these cases IL5 and IL10 production and the Th2 immune response prevail 207 and subjacent causes of immunosuppression should be investigated 209 The granulomas affecting these patients are poorly organized oedematous rich in polymorphonuclear leucocytes and usually have central suppuration and coagulation necrosis The number of fungal cells in these patients is high with many cells multiplying and many cells of minute size Figs 7C 8A Fig 7 A Paracoccidioidomycosis infiltrated erythematous and well demarcated lesions on face B Paracoccidioidomycosis compact granuloma with epithelioid and giant cells A fungal cell is well observed in the cytoplasm of a giant cell HE x40 Verrucous lesions are generally observed in patients with more balanced hostparasite equilibrium a low to moderate number of cutaneous lesions and good clinical condition Verrucous lesions correspond to a type of tissue response characterized by marked pseudoepitheliomatous hyperplasia Fig 8C This type of lesion requires clinical and histological differentiation from squamous cell carcinomas The presence of inflammatory granulomatous infiltration accompanied by fungal cells in the dermal tissues may help makes this distinction clear however the risk of misdiagnosis increases when superficial or shave biopsies are performed 244 The Open Microbiology Journal 2017 Volume 11 Mendes et al Fig 8 A Paracoccidioidomycosis ulcerated lesion with hemorrhagic dots and elevated borders on the face B Paracoccidioidomycosis fungal cells stained in black some in active multiplication GrocottGomori x 40 C paracoccidioidomycosis marked pseudoepitheliomatous hyperplasia with dense inflammatory infiltration in the dermis Fungal cells can be observed HE x10 Mucosal lesions in PCM patients are characterized by superficial ulcers with microgranulation and haemorrhagic pinpoints often referred to as mulberrylike stomatitis These mucosal lesions also exhibit infiltrated borders or infiltrative tissue at their base This clinical feature is often observed in patients with lesions of the buccal ocular or genital mucosa The histological features of mucosal lesions are similar to those of ulcerated cutaneous lesions Methenamine silver nitrate GrocottGomori stain and periodicacid of Schiff PAS stains are frequently used to better detect the presence of Paracoccidioides in dermal tissues Fig 8B 10113 Lymph Nodes Involvement of the submandibular lymph nodes was first reported by Lutz 1 P brasiliensis lymphotropism was suggested by Haberfeld 1919 210 and Niño 1939 pointed to a direct relationship between poor prognosis and the early appearance and severity of lymphadenopathy 211 The relevance of lymph node involvement may be assessed based on its frequency in clinical and autopsy studies the detection of subclinical involvement the alterations of the lymphatic system identified by lymphographic and scintigraphic evaluations and more particularly the depression of the cellmediated immune response resulting from lymphoid tissue damage Paracoccidioides sp may spread to the lymph nodes via the haematogenous or lymphatic routes The fungus is drained from organ lesions to the regional lymph nodes and then spreads to other lymph nodes via the lymphatic system The haematogenous route allows the fungus to spread to lymph nodes through the arteries that feed them Subclinical lymphadenopathy defined by the detection of paracoccidioidallesions in lymph nodes that are considered normal on clinical examination was found in the lymph nodes that drain affected areas as well as in others quite distant from fungal lesions 212 214 The latter situation is admittedly caused by haematogenous spread Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 245 Lymph node enlargement may be the main clinical complaint being the rule among children adolescents and young adults who exhibit the acutesubacute form of PCM also known as juvenile form 26 195 198 199 215 217 The lymphatic chains most often affected are those of the head followed by the supraclavicular and axillary nodes 212 In the head the submandibular and anterior and posterior cervical nodes are most frequently involved The submental tonsillar pre and postauricular and even the suboccipital lymph nodes may also be affected with variable frequency Although rare intercostal epitrochlear and popliteal lymph node involvement was described primarily in severe cases Abdominal lymphadenopathy originally described in 1915 218 219 has been frequently reported in the Centre West region of Brazil as well as in the Botucatu area 220 221 occasionally with clinical manifestations that mimic acute abdomen 219 222 The presence of large tumourlike masses on palpation is suggestive of lymphoproliferative disease Abdominal lymph node enlargement can cause extrinsic compression Obstructive jaundice is not uncommon among patients with involvement of the hepatic hilum lymph nodes and compression of the extrahepatic bile ducts 223 224 In addition inferior vena cava syndrome was described in a patient with PCM and abdominal lymphadenopathy 225 Mesenteric lymphadenopathy can lead to malabsorption occasionally attended by chylous ascites 226 228 Involvement of the deep lymphatic system the location of which makes difficult its characterisation on physical examination can be assessed by ultrasound 229 computerised axial tomography CAT 230 lymphography 229 231 234 or lymphoscintigraphy 235 Patients with the chronic form of PCM may exhibit cervical and submandibular lymphadenopathy in relation with the drainage of lesions of the mucous membrane of the UADT However it is worth noting that patients with the PCM chronic form and without lymphadenopathy on physical examination were found to exhibit severe involvement of the deep lymphatic system on bipedal lymphography 234 The clinical characterisation of lymphatic involvement is overall difficult because different lymph nodes from various chains may be affected and exhibit different characteristics in the same patient For this reason lymphatic involvement is clinically classified into three types based on the largest diameter of the lymph nodes and the presence or not of suppuration as follows 1 inflammatory nonsuppurative the largest diameter of all lymph nodes is less than 20cm and none exhibits suppuration 2 tumoural no lymph node exhibits suppuration and the diameter of at least one is 20cm and c suppurative at least one lymph node exhibits fluctuation or fistula independently from its diameter Fig 9A The lymph nodes of patients with the inflammatory nonsuppurative type tend to be painless noncoalescent and mobile without exhibiting either heat or redness In patients with tumoural lymphadenopathy the lymph nodes are usually painful on palpation fixed to deep or superficial planes and coalescent with redness andor heat 236 It is worth noting that when classifying the type of lymphadenopathy the established type applies only to the time of assessment if patients do not receive appropriate treatment or at times in spite of it the infection may progress and the lymph nodes may become even larger andor suppurate Bipedal lymphography affords an excellent morphological assessment of the lymphatic system The lymphangiographic phase shows dilation segmentation delay in outflow and less often obstruction of the lymphatic vessels The lymphographic phase reveals abnormalities in filling shape size form of presentation and number of opacified lymph nodes 234 Patients with the chronic form of PCM exhibit symmetrical lymphographic alterations 234 Lymphoscintigraphy affords an excellent functional assessment of the lymphatic system This test allows the investigation of the lymphatic flow by means of semiquantitative and quantitative variables and lymph node uptake of the radiotracer 235 In patients with the acutesubacute form of PCM the lymphatic flow is increased in the lower limbs before antifungal treatment The low serum albumin levels exhibited by this population of patients might account for this finding In this population the flow pattern does not change after the first few months of treatment in contrast patients with the chronic form of PCM exhibit increased lymphatic flow after the onset of treatment 235 102 Otolaryngological Aspects The involvement of the mucous membrane of the upper aerodigestive tract UADT ie the nasal fossa oral cavity oropharynx hypopharynx and larynx is highly relevant due to its high frequency and the ease of collecting samples for the identification of the aetiological agent 237 241 246 The Open Microbiology Journal 2017 Volume 11 Mendes et al Aguiar Pupo 1936 conducted the first systematic study of lesions in mucous membranes caused by P brasiliensis and described moriform ulcerative stomatitis which later on was given his name 237 Hoarseness pain and difficulty swallowing burning in the throat the feeling of bulging or a wound in the mouth and dyspnoea are the most common clinical manifestations 241 The lesions in the mucous membrane might be very painful especially when eating hot or very salty foods In general more than one UADT area is involved with most lesions being in the larynx followed by the oropharynx hypopharynx and oral cavity which exhibit similar prevalences Bilaterality is almost always observed for all sites but the morphology of the lesions is highly variable Hyperaemia moriform lesions swelling granulomatous infiltrative lesions ulcerations granular lesions infiltrative lesions and vegetating lesions have all been described The progression of Aguiar Pupos moriform ulcerative stomatitis is slow and its presence is suggestive of PCM This form is characterised by an ulcerated area the base of which has a fine granular mulberrylike appearance Together with hyperaemia it is the type of lesion that predominates in the oral cavity In the oropharynx lesions affect more frequently the soft palate and anterior and posterior pillars followed by the lateral and posterior walls uvula tongue and tonsil area Hypopharyngeal lesions are distributeduniformly among the lateral anterior and posterior walls and the pyriform sinus and hyperaemia and moriform lesions are the predominant lesion types All the areas of the larynx may be affected more frequently the ventricular fold arytenoid area vocal cords and the free portion of the epiglottis followed by the laryngeal surface of the epiglottis aryepiglottic fold subglottic area and the laryngealventricle space Swelling granular lesions hyperaemia and moriform lesion are the most common lesion types having similar prevalences Vegetating lesions and ulcers are found in very few cases Involvement of the nasal mucous membrane columella and nasal septum has also been reported The gums are frequently involved accompanied by teeth loosening These findings are suggestive of PCM make eating difficult and havea negative impact on the patients nutritional status 238 241 Lastly hard palate perforation must be mentioned although it is a rare occurrence 242 The functional study of laryngeal sequelae in PCM patients revealed frequent and severe dysphonia dysphonia index 70 characterized by roughness and breathness The Dr Speech Tiger Electronics analysis program did not accept five out of 15 voices due to severe dysphonia Jitter was elevated in five patients 243 The severe dysphonia observed in patients with laryngeal sequelae may have important social consequences for PCM patients 103 Adrenal Glands Adrenal involvement by P brasiliensis was first reported by Viana 1913 244 1914 245 during the autopsy of a patient with disseminated disease it was subsequently reported in a patient with areas of pulmonary fibrosis 246 It was in 1952 that the signs and symptoms exhibited by PCM patients were correlated with clinical manifestations of chronic adrenal insufficiency 247 The Thorn test for assessment of the adrenal function was applied to PCM nine years later 248 This test revealed a high incidence of adrenal involvement which thus came to be considered as the third most frequent location of disease and that the adrenal reserve was reduced in 48 of the assessed patients 248 The tropism of P brasiliensis for the adrenal glands might be explained by the local reduction of the cellmediated immunity caused by its high glucocorticoid concentrations 249 The main signs and symptoms of chronic adrenal insufficiency in patients with PCM are malaise fatigue anorexia weight loss arterial hypotension orthostatic hypotension hyperpigmentation of the skin and mucous membranes nausea vomiting and reduced libido and sexual potency Hyperpigmentation is usually reported or confirmed by patients and is most evident on the oral mucosa nipples penis areas exposed to friction such as the elbows and scars The serum potassium calcium and urea levels are usually elevated and the sodium and chloride levels decreased The diagnosis of adrenal insufficiency is established based on urinary 17hydroxycorticosteroid levels and plasma cortisol levels before and after adrenal stimulation with semisynthetic adrenocorticotropic hormone ACTH The baseline levels are low and the response to stimulation is insufficient or absent 248 250 Evaluation of serum aldosterone levels before and after adrenal stimulation with semisynthetic ACTH showed low levels before stimulation in several patients and an absence of response to ACTH in some cases 250 The plasma ACTH levels are high in patients with PCM and clinical manifestations compatible with Addisons disease 251 suggesting that the measurement of ACTH might be useful for early diagnosis of chronic adrenal Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 247 insufficiency Diagnostic imaging is a significant contribution to the identification of adrenal involvement in PCM On computerized tomography the adrenals exhibit irregular contours as well as volume and density abnormalities Ultrasound allows for assessing the shape contours density and size of the adrenal glands Comparison of findings on computerized tomography and ultrasound with the plasma cortisol and aldosterone levels before and after stimulation with ACTH revealed limited adrenal reserve in 53 of patients abnormalities on computerized tomography in 43 and ultrasound abnormalities in 17 251 The combination of both imaging methods allowed establishing a diagnosis of adrenal involvement in 85 of cases 251 The adrenal function seldom recovers after antifungal treatment for PCM but persistence of residual adrenal insufficiency is much more frequent 252 104 Digestive System Involvement of the gastrointestinal tract was reported in the earliest studies of patients with PCM One of the cases reported by Lutz exhibited chronic diarrhoea the aetiology of which was not established 1 Viana described a case of disseminated PCM and the autopsy revealed mycotic ulcerations in the ileum appendix and colon The introduction of new techniques for the investigation of the gastrointestinal tract led to more reports of new cases although studies with large case series remain scarce and studies assessing the full extension of the gastrointestinal tract are even rarer 253 256 Gastrointestinal complaints are reported by more than 50 of patients when interrogation targets the digestive system In such cases sialorrhoea dysphagia halitosis abdominal pain bloated feeling pyrosis and abnormalities in intestinal motility are frequent findings followed by regurgitation vomiting hiccups and presence of an abdominal mass 256 In another study the most frequent clinical manifestations were abdominal pain changes in bowel habits nausea and vomiting 255 Abdominal pain is usually of the colic type alone or alternating with continuous pain Diarrhoea lasts more than 15 days and usually consists of two to six liquid or pasty stools per day Some patients report the presence of blood streaks or mucus in the stools Constipation is as frequent as diarrhoea lasting up to 10 days and usually being due to extrinsic compression or isolated intestinal lesions Constipation may also be associated with severe obstructive disorders for this reason patients require careful follow up 255 Some patients undergo periods of alternating constipation and diarrhoea It should be noted that gastrointestinal symptoms occur more frequently among patients with the acutesubacute form of the disease and clinical evidence of involvement of the abdominal lymphatic system which is characterised by palpable masses 255 Gastrointestinal manifestations may be the earliest complaints among such patients Radiological investigation of the gastrointestinal tract reveals anatomical or functional abnormalities in 89 of patients In most cases such abnormalities involve more than one segment being most frequent in the ileum stomach duodenum jejunum and ascending and descending colon Involvement of the oesophagus and rectum is rare and involvement of the appendix is extremely rare 256 Functional disorders are more common than the anatomical disorders Among the former hypersecretion hypotonia reduced peristalsis and barium column flocculation are the most frequent The anatomical abnormalities with the highest incidence include mucosal fold thickening dilation extrinsic compression by the liver spleen or lymph nodes stenosis and stiffness 256 Some patients progress into intestinal obstruction or subobstruction and consequently into acute abdomen that requires surgical treatment Some patients exhibit proteinlosing enteropathy 227 257 258 and deficient absorption of glucose 226 227 257 and more commonly of fat 226 227 255 257 Although obstruction of the lymphatic vessels is the essential cause of these phenomena their clinical manifestations in the gastrointestinal tract justify their inclusion in this section These disorders are common among patients with major involvement of the abdominal lymphatic system Diarrhoea chylous ascites hypoalbuminaemia and lymphocytopaenia are characteristic of proteinlosing enteropathy Lymphatic stasis due to abdominal lymphadenopathy might cause hypertension of the lymphatic system with consequent extravasation of the protein and lymphocyterich lymph into the intestinal lumen The mucosal ulcerations caused by P brasiliensis may contribute to the protein loss The presence of diarrhoea is not necessary for protein loss to occur a factor that should always be considered whenever a patient exhibits considerable reduction of 248 The Open Microbiology Journal 2017 Volume 11 Mendes et al serum albumin in the absence of renal loss or impaired synthesis Neither clinical nor radiological assessment is able to demonstrate the occurrence of proteinlosing enteropathy For this reason the faecal excretion of 51Crlabelled albumin should be investigated Some patients exhibit extremely fetid stools diarrhoea steatorrhoea chylous ascites and abnormal results in the plasma turbidity test after fat overload these findings are characterised by flattened curves and increased faecal fat Such patients often exhibit radiological intestinal abnormalities primarily in the ileocaecal region Carbohydrate absorption which is independent from the lymphatic system and takes place at more proximal regions of the small intestine is less frequently and less severely affected compared to fat absorption and protein loss The investigation of Dxylose absorption allows the establishment of a diagnosis of carbohydrate malabsorption Few studies have investigated liver involvement in PCM 223 259 261 The presence of liver enlargement that decreases under antifungal treatment points to PCM especially when the tropism of P brasiliensis for the mononuclear phagocyte system is taken into consideration In general liver lesions are not associated with clinical manifestations although one case with intense jaundice signs and symptoms of severe liver failure and terminal coma was reported 260 Some patients exhibit jaundice which is due to extrinsic compression of the bile ducts by enlarged hepatic hilum lymph nodes 223 259 Liver biopsy may reveal lesions with variable intensity from mild and unspecific to severe consisting of portal and intrasinusoidal granulomas No patient analysed in this study exhibited signs of portal hypertension 261 A recent study that assessed the liver and bile ducts using radioisotopes found intrahepatic cholestasis bile duct obstruction single or multiple focal defects and heterogeneous hepatic uptake Intrahepatic cholestasis was most frequent among patients with the acutesubacute form of PCM 262 Pancreatic PCM which may mimic neoplasms of the pancreatic head or abdominal tumours has been reported despite being rare 263 266 One patient had a history of severe weight loss weakness dizziness general malaise bloating and intense itch attended by jaundice choluria and faecal acholia The material obtained through computerized tomographyguided puncture aspiration of the pancreatic head contained pancreatic epithelial cells and several P brasiliensis yeastlike cells 266 105 Bones and Joints Knowledge of the involvement of bones and joints in PCM was obtained in case reports or small case series few prospective and systematic studies have been conducted 267 268 Such studies have reported a frequency of involvement between 16 and 20 A review of studies on bone involvement published until 1964 is also available 269 In general P brasiliensis spreads to bones via the haematogenous route which explains its presence in patients with disseminated disease 268 The isolation of fungi from blood cultures supports this view 269 In addition analysis of some cases suggests that bone involvement might also occur from lesions in adjacent tissues In turn joint affection might arise from preexisting lesions in one or more of the bones that compose the joint 270 271 However personal observation of a patient with joint involvement but without radiological evidence of bone affection suggests that haematogenous or lymphatic spread is a possibility Bone lesions begin in the medullary layer extending first to the cortex and then to the periosteum 272 The lesions are usually asymptomatic and when they affect superficial bones they might be visible or palpable in all other cases imaging methods are necessary to detect them By contrast joint affection has exuberant clinical manifestations pain and functional impotence with increased volume and temperature on physical examination Although any bone might be affected lesions predominate in the chest ribs and sternum shoulder girdle clavicles and scapulae and upper limbs 257 269 These locations easily visualised on chest radiographs are very helpful for the differential diagnosis between lunginvolved PCM and tuberculosis The presence of bone lesions in the chest shoulder girdle or upper limbs reinforces a diagnostic hypothesis of PCM In general radiographs show lytic lesions without perifocal reaction mild or no periosteal reaction and well defined margins The cortical bone is destroyed in almost half of cases Joint involvement is detected in about onethird of cases with bone involvement 267 Skeletal scintigraphy with 99mTcmethylene diphosphonate MDP is a highly valuable technique for the detection Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 249 of PCM lesions due to its high sensitivity the early appearance of abnormalities assessment of the full skeleton in a single exam rare contraindications noninvasiveness and return to normal conditions after treatment 273 The treatment of PCM is attended by fibrosis and bone neoformation with consequent changes in the characteristics of lesions progression is very gradual 106 Bone Marrow Bone marrow involvement usually occurs in patients with the acutesubacute form of PCM being rare in cases with the chronic form Bone marrow biopsy provides the best samples to demonstrate the hostfungus interaction 274 275 The lesions are variable from focal and compact to diffuse and loose Reticulin fibrosis predominates in the most localised lesions while coagulative fibrosis prevails and reticulin fibrosis is discrete in more extensive and looser lesions Residual haematopoiesis is impaired in the cases with more extensive lesions Bone marrow involvement may contribute to the occurrence of anaemia leukopaenia and thrombocytopaenia as well as to the absence of lymphocytosis and monocytosis in the peripheral blood Lastly it should be noted that the presence of a leukoerythroblastic reaction in the peripheral blood is the blood abnormality that best points to bone marrow affection by P brasiliensis 274 275 107 Central Nervous System CNS The occurrence of seizures in a patient with disseminated PCM skin lesions was the first clue pointing to possible CNS involvement 276 Subsequent publication of several case reports showed that this disorder is more common than previously thought The frequency of CNS involvement varies considerably according to the methods used for investigation Complete autopsies are not always performed especially as concerns the spinal cord and usually tend to correspond to terminal cases with extensive dissemination of disease Clinical studies in turn do not always include neurological assessment especially in relation to auxiliary diagnostic tests as many patients with CNS involvement exhibit very discrete or no symptoms One prospective study that targeted the CNS revealed symptoms suggestive of disease in 25 of the cases 277 In general clinical manifestations of CNS involvement occur in patients with previous or ongoing involvement of the organs that are more frequent targets of PCM however cases with CNS involvement alone have been reported 278 Lesions may be located in the neural parenchyma or the meninges giving rise to two polar forms parenchymatous or pseudotumoural which is most frequent and meningeal 279 In one study the clinical manifestations were classified as pseudotumoural in 24 of 34 analysed cases Eleven of these cases exhibited multiple granulomas meningoencephalitic 734 or meningitic 334 279 lesions were found in the brain 1324 cerebellum 624 or both 524 279 Meningeal involvement may be diffuse or localised and most often affects the base of the brain The development of disease is usually insidious and may be confounded with tuberculous meningoencephalitis Inflammation might cause severe intracranial hypertension Cerebrospinal fluid CSF abnormalities are unspecific and include mild moderate or intense pleocytosis with predominance of lymphocytes and increased protein levels with predominance ofγglobulin attended by decreased CSF glucose The isolation of P brasiliensis from the CSF is extremely rare The clinical manifestations of the parenchymatous or pseudotumoural form are highly variable as a function of the number size and localisation of granulomas Symptoms of intracranial hypertension predominate which appear progressively with signs of localisation characterised by motor or sensory deficits language disorders and cerebellar ataxia Focal or generalised seizures and papilloedema have also been reported 279 Findings on computerized axial tomography CAT and magnetic resonance imaging MRI are not pathognomonic CAT shows ringenhancing round lesions of variable localisations with no signs of bone neoformation or destruction little perifocal oedema and a discrete compression effect 280 Assessment by MRI is better compared to CAT particularly for lesions in the posterior fossa and when paramagnetic contrast agents are used 281 The lesions are characterised by an iso or hypointense T1 signal a hypointense T2 signal peripheral oedema and a nodular or ring contrast enhancement 281 The authors of this MRI study found a correlation between hypointense T2 signal and a chronic granulomatous process Tests performed after treatment revealed disappearance of perilesional oedema but persistence of the hypointense lesions on the T2weighted scans 250 The Open Microbiology Journal 2017 Volume 11 Mendes et al Few cases of spinal cord involvement were reported perhaps because this region is not routinely assessed on autopsy Patients may exhibit progressive manifestations including paraesthesia anaesthesia and weakness in the lower limbs faecal and urinary incontinency and neurogenic bladder presenting with episodes of urinary retention 282 Early diagnosis of neuroPCM is based on the identification of lesions in the organs that are the more frequent targets of PCM andor the presence of epidemiological antecedents denoting high risk of infection with P brasiliensis in patients with neurological complaints 108 Urogenital System Few cases of PCM with urogenital involvement have been reported and the number of published case series is even smaller Viana 1914 found kidney lesions during the autopsy of a patient with disseminated disease 245 Urogenital lesions usually occur in cases in which other organs are also affected are almost exclusive to males and are seldom responsible for the patients main complaints Rather such lesions tend to be incidental autopsy findings 283 Themost often affected structures are the epididymis testicles and prostate alone or in combination 284 Pain in and increased volume and consistency of testicles and epididymis difficulty urinating pollakiuria and enlarged hardened prostate are found in cases with urogenital PCM Urogenital involvement is rare among females representing only 10 of cases Despite the low frequency cases with lesions in the ovaries and adnexa 285 placenta 286 287 and breasts 288 290 have been reported 109 Thyroid Very few cases of thyroid affection by P brasiliensis have been reported being incidental autopsy findings 160 291 One single case of symptomatic thyroid involvement has been described the patient exhibited a chronic condition characterised by weight loss nervousness and neck pain followed by restlessness irritability anxiety insomnia and excessive sweating The patient also reported neck pain radiating to the ear 292 Physical examination revealed thyroid hypertrophy and erythema on the skin over the gland The sample collected via puncture aspiration contained typical follicular cells and the characteristic P brasiliensis yeastlike cells Chest radiographs revealed lung involvement The first study of the thyroid function in patients with PCM assessed the serum thyroxin T4 and triiodothyronine T3 levels and the response to thyrotropinreleasing hormone TRH The results showed that the serum T3 levels were low in a large number of patients all of whom exhibited severe forms of PCM These findings suggest that the peripheral conversion of T4 into T3 is reduced but do not point to the occurrence of any type of hypothyroidism ie primary secondary or tertiary 293 1010 Eyes and Adnexa The first report of eye affection by P brasiliensis dates to 1923 276 and until 1988 approximately 50 cases were reported corresponding to patients with lesions in other organs 294 Only one of the eyes is affected with no predominance of either Palpebral and conjunctival lesions are very frequent while there are few reports of anterior uveitis or choroiditis 294 295 Palpebral lesions begin as papules usually close to the lid margin that then grow and develop ulcers in their centre The ulcers base exhibits fine haemorrhagic points and thickened hardened margins evoking the moriform lesions described by Aguiar Pupo The earliest eye lesions may mimic styes hordeolum or even bacterial blepharitis 294 1011 Other Organs P brasiliensis can involve any organ giving rise to symptomatic or asymptomatic lesions the latter are usually casual or incidental autopsy findings The involvement of some organs is so rare that PCM is not even suspected except when attended by lesions in more common sites It was on these grounds that heart vessel pituitary spleen and striated muscle involvement was detected 296 It should be noted that the modern diagnostic imaging methods and performance of more invasive diagnostic procedures increased the frequency of confirmation of lesions in these organs 11 CLASSIFICATION OF CLINICAL FORMS The hostfungus interaction can be characterised as infection or as disease with several clinical forms This classification is based on criteria formulated by a group of specialists who met at the Third International Congress on Paracoccidioidomycosis held in Medellin Colombia 297 with some modifications resulting from the study of the acutesubacute form 217 the introduction of the regressive form wellestablished for other systemic Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 251 mycoses a definition of the mixed 298 and the isolated organic forms 299 and the characterisation of severity 219 220 Paracoccidioides infection is exhibited by healthy individuals who contacted the fungus and developed an efficacious cellmediated immune response that was able to hinder the progression of infection into actual disease Infection is confirmed by a positive intradermal reaction to the specific antigen or identification of latent foci in the autopsy of individuals who died from other causes 83 The regressive form is the most benign type of PCM patients only exhibit mild clinical manifestations usually involving the lungs positive skin reaction to paracoccidioidin and clinical regression even without treatment 302 303 This form is seldom diagnosed because a lack of awareness of P brasiliensiss ecological niche does not allow correlating a suspicious contact with selflimited clinical manifestations which are thus attributed to other causes The acutesubacute chronic mixed and isolated organic forms represent progressive disease and are characterised by signs and symptoms associated with the involvement of one or more organs The characterisation of these forms is based on the patients age duration of symptoms clinical manifestations the presence of associated diseases and aggravating factors the overall state of health and nutritional status plain chest radiographs and skin reaction to paracoccidioidin and serum antiP brasiliensis antibodies as measured by the double immunodiffusion DID test The acutesubacute form AF of PCM usually affects children adolescents and young adults for which reason it is also known as juvenile form The symptomatology develops over a short period of time with a median of two months 198 It is characterised by clinical manifestations that are compatible with involvement of the mononuclear phagocyte system to wit lymph node enlargement hepatomegaly andor splenomegaly and less often bone marrow involvement Lymphadenopathy occurs in several superficial andor deep lymphatic chains and is the predominant clinical manifestation of disease The involvement of mucous membranes is infrequent occurring in 17 to 20 of cases and pulmonary involvement is even rarer being present in 5 to 10 of patients 26 198 304 P brasiliensis may be isolated from the bronchoalveolar lavage fluid of patients with AF with no clinical or radiological evidence of lung involvement 305 In such cases the lungs merely act as portal of entry According to the predominant manifestations AF can be subdivided into four clinical forms 213 a with superficial lymphadenopathy b with abdominal or gastrointestinal involvement c with bone involvement and d with other clinical manifestations The identification of adult patients with PCM and clinical characteristics of AF is not a rare event In such cases the clinical form should be defined as acutesubacute also known as juvenile type 297 A recent study showed that patients under 30 years of age and with the AF of PCM exhibit a higher incidence of skin lesions more frequent and more severe eosinophilia and higher serum levels of precipitating antibodies as assessed by the DID test compared to patients over 29 199 These differences allow establishing a clinicallaboratory pattern of AF that manifests in children adolescents and young adults and another pattern occurring in adults 199 For the purpose of decisionmaking on treatment and prognostic assessment AF may be classified as moderate or severe The possibility of mild disease is never considered for this population of patients because the early and rapid development of disease and the intense involvement of the mononuclear phagocyte system are indicative of severe depression of the cellmediated immune response The chronic form CF of PCM usually manifests in adults over 30 years of age and with longlasting symptoms ie for more than six months Lung involvement is the rule although it may be absent in some cases and involvement of the mucous membranes of the UADT is very frequent While lymphadenopathy also occurs it usually involves lymphatic chains of the neck in a localised manner only and is not the dominant finding As to its severity CF is classified as mild moderate or severe Patients with mild CF are in good state of health and exhibit a satisfactory nutritional status and weight loss is less than 5 of the patients usual body weight Lung involvement which is very frequent in CF is mild or may even be absent Tegumentary involvement especially the mucous membranes of the UADT is discrete or absent When present lymphadenopathy is limited to the chains of the head and is of the inflammatory nonsuppurative type Patients do not exhibit signs of involvement of other organs or systems The serum levels of antiP brasiliensis antibodies are low and the intradermal reaction to paracoccidioidin is strong Lastly all of the suggested criteria should be met for CF to be classified as mild 252 The Open Microbiology Journal 2017 Volume 11 Mendes et al The other pole is represented by severe CF characterised by poor overall state of health and nutritional status with weight loss greater than 10 of the usual body weight Patients exhibit intense respiratory symptoms and chest radiographs evidence lung involvement When present lymphadenopathy is not limited to the chains in the neck and is of the tumoural or suppurative type Skin lesions are usually present and are severe Involvement of other organs such as the adrenal glands and CNS is frequent In general patients exhibit high levels of antiP brasiliensis antibodies in the serum and negative reaction on the paracoccidioidin skin test The presence of three of these criteria suffices to characterise the severe CF of PCM The moderate CF of PCM is intermediate between the polar forms just described In general patients have a moderately affected overall state of health and nutritional status with weight loss equivalent to 510 of the usual body weight Signs of involvement of other organs or systems such as the adrenal glands CNS gastrointestinal tract and bones are usually absent The serum levels of specific antibodies are moderately elevated and the response to the intradermal test with paracoccidioidin is also moderate The population of patients with this form of PCM is highly heterogeneous Some patients meet almost all of the criteria for the mild form of CF thus representing a group with moderate CF that is quite close to the mild form Consequently these patients may be considered as presenting a mildtomoderate form of disease Other patients meet just one or two of the criteria for severe CF thus representing a group with moderate CF that is close to the severe form and correspondingly can be classified as a moderatetosevere form Lastly in some patients the severity of the clinical manifestations is equally distant from both the mild and severe forms thus these patients are simply classified with the moderate form of disease 111 Mixed Forms There are patients presenting some clinical manifestations that are typical of the AF while other findings are proper to the CF which makes their classification difficult These variations of the clinical presentation of PCM should be considered as mixed forms and are seen in patients with severe depression of the cellmediated immune response and extensive dissemination of disease it was for this population of patients that the term mixed forms was first suggested 298 112 Isolated Organic Form In some rare cases the clinical manifestations of PCM relate to one single organ but do not meet the criteria for either AF or CF In these cases diagnosis usually requires invasive methods and histopathological assessment which often reveals involvement of some contiguous structures most often the lymph nodes These patients should be classified as with the isolated organic form of disease a term already used by pathologists 299 113 Residual Forms Sequelae are very frequent in PCM because a considerable portion of patients develop sequelae Pulmonary sequelae stand out due to their frequency severity and the limitations they impose on the patients lives and are primarily characterised by fibrosis and emphysema Even when treatment is appropriate patients with Addisons syndrome frequently require hormone replacement therapy for life The neurological sequelae vary greatly according to the localisation of lesions but generally impose considerable limitations to the patients activity Tracheal lesions cause hardtotreat sequelae which occasionally require surgical intervention Lesions in the gastrointestinal tract might cause obstruction or subobstruction requiring twostage surgical correction Skin and mucosal lesions are very often disfiguring especially laryngeal lesions causing considerable and often irreversible impairment of the patients voice Other organs may also develop sequelae 12 CLINICAL MANIFESTATIONS OF PCM CAUSED BY P LUTZII On the contrary of laboratory evaluation studies on clinical manifestations are scarce in PCM caused by P lutzii with only two publications two patients with chronic form 300 and one case of fatal fungemia 301 A comparison of the clinical manifestations between patients with PCM caused by P brasiliensis and cases by P lutzii is ongoing in the Faculdade de Medicina Universidade Federal de Mato Grosso do Sul Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 253 13 CLINICAL MANIFESTATIONS IN SPECIAL HOSTS 131 AIDSPCM CoInfection After the emergence of the AIDS pandemic in the 1980s cases of comorbidity PCM and HIV began to be reported in PCM endemic countries particularly Brazil Venezuela and Colombia 298 306 Different from histoplasmosis and cryptococcosis which are more commonly identified in AIDS patients large PCM case series have shown that in Brazil only 45 of patients with PCM have comorbidity HIV infection 307 309 and approximately 15 of AIDS cases have comorbidity PCM 307 310 This relatively low prevalence has been attributed to the routine use of cotrimoxazole for Pneumocystis jirovecii pneumonia PJP prophylaxis in patients with AIDS which may also prevent PCM 307 However Morejón et al 307 reported that a high percentage of patients with comorbid PCMHIV regularly used cotrimoxazole prophylaxis for the prevention of PJP Additionally the epidemiological factors associated with fungi exposure could explain the low observed prevalence of ParacoccidioidesHIV coinfection AIDS has been identified as more prevalent in urban centres while PCM generally predominates in small towns with rural economies therefore urban AIDS patients may have less exposure to Paracoccidioides sp In our previous study in which we evaluated the prevalence of paracoccidioidical infection in HIVinfected individuals using the gp43 intradermal test we found that 122 of HIV patients were coinfected with Paracoccidioides sp 311 and were therefore at risk of becoming ill due to the decreased cellular immunity that is characteristic of HIV infection This prevalence was lower than that observed in rural areas of the same region with a coinfection prevalence of 47 previously identified among rural settlers 91 Patients with the PCMHIV comorbidity have been reported to be younger and less involved with farming activities 306 307 and the vast majority of these patients have CD4 counts 200cellsmm 3 The clinical manifestations of PCM in HIV patients range from mild to severe PCM cases with extrapulmonary organ involvement including involvement of the lymph nodes liver and spleen have been more frequently identified in HIVpositive than HIV negative patients 307 Involvement of the mononuclear phagocyte system is characteristic of acutesubacute PCM however in patients with chronic PCM the lungs and mucous membranes of the upper aerodigestive tract have also been found to be affected 298 307 These findings suggest that these patients have a mixed form of PCM which was included in the PCM classification proposed in 1987 312 Regarding the diagnosis of PCM attention should be paid to the fact that antibody detection tests have demonstrated reduced sensitivity in cases with HIV 307 Therefore one should not completely rule out a diagnosis of PCM in HIV patients when serological test results are negative In these cases microbiological methods should be preferentially used as diagnostic tests such as experimental antigenbased and molecular biology tests are not yet available for use in routine care The combination of two methods of antibody detection ELISA tests and either counter immunoelectrophoresis or immunodiffusion has shown to have increased sensitivity 313 Antifungal treatment recommendations do not differ by HIV status however itraconazole treatment should be avoided in cases with tuberculosis because this antifungal agent may interact with rifampicin thereby reducing serum itraconazole levels Tuberculosis coinfection has previously been observed in 94 to 583 of cases with PCMHIV comorbidity 307 310 Secondary prophylaxis with antifungal agents until T CD4 lymphocytes levels reach at least 200cellsmm 3 has been suggested based on studies assessing other opportunistic mycoses 177 132 PCMLymphoproliferative Disorders Studies on the association between PCM and malignant tumours are relatively scarce Carcinomas are the most frequently diagnosed type of neoplasia in PCM patients that has been attributed to the humoral and cellular immune dysregulation induced by P brasiliensis 314 On the other hand the immunesupression caused by some neoplasias andor their treatments could reactivate quiescent P brasiliensis foci Although the comorbidity PCMlymphoma is rare with cases showing PCM previously to lymphoma it is possible to suggest that the chronic stimulation by paracoccidioidal antigen could be implicated in the origen of B lymphomas 314 Additional studies should be performed to better understand this subject 14 LABORATORY FINDINGS There are few studies on the laboratory abnormalities exhibited by patients with PCM upon admission and during 254 The Open Microbiology Journal 2017 Volume 11 Mendes et al antifungal treatment and they generally involve small numbers of patients and a short followup period The abnormalities detected upon admission ie before the onset of antifungal treatment reflect the effects of PCM and must return to normal In turn the abnormalities that appear after the onset of treatment are usually side effects 141 Complete Blood Count This test usually shows normocytic and normochromic anaemia Leukocytosis might be present most often in cases with CF Eosinophilia is the most common finding being most frequent among cases of AF The erythrocyte sedimentation rate is elevated in the vast majority of cases and serves as an auxiliary criterion of cure 315 317 142 Hepatobiliary System Abnormalities of hepatobiliary variables are more intense in AF compared to CF Table 3 318 These studies confirm the results of previous studies conducted with small number of patients with AF 217 319 143 Metabolic Abnormalities and Acute Phase Reactants The changes most frequently found in both AF and CF are reductionsin serum cholesterol and albumin levels and elevated levels of α1acid glycoprotein globulins and mucoproteins The reduction of serum cholesterol and albumin and elevation of globulin levels are more intense in AF compared to CF Table 3 The elevation of γglobulin mucoproteins and α1acid glycoprotein were previously reported 320 Table 3 Prevalence and intensity of the altered biochemical variables in 200 paracocidioidomycosis patients with active disease before treatment with antifungal compounds Comparison between the acutesubacute and the chronic form Prevalence Intensity Variables AFCF AF CF p value AF CF p value AST a 127 217 96 005p010 2516 1506 013 ALT a 182 239 163 010 2816 1706 006 TB a 62 87 53 020 6446 1912 007 CB a 120 217 85 005 105143 5051 022 ALP a 260 500 178 0001 2820 1404 0001 γGT a 326 478 247 002 5266 2729 002 Triglycerides a 107 44 127 012 220170 137050 Glucose a 149 152 148 094 123032 132041 059 Glucose d 43 65 35 039 070019 075034 Cholesterol a 337 188 387 001 126037 116017 018 Cholesterol d 663 813 613 001 072013 081013 0001 Total lipids d 88 235 39 001 082007 082012 Calcium a 87 93 85 087 104004 104004 Calcium d 71 116 57 018 095004 095003 071 Uric acid d 119 30 154 007 113012 Phosphorus a 143 143 143 10 106002 118018 018 Phosphorus d 40 400 375 079 081010 086010 016 α1 glycoprotein a 378 389 375 092 160051 139037 024 Total protein a 316 489 257 0003 108008 110030 072 Total protein d 48 43 50 084 085003 080029 Albumind 242 304 202 026 074021 083012 0001 Globulins a 730 844 693 0046 144031 123020 0001 Mucoproteins a 527 565 515 068 160050 15843 091 AF acute subacute form CF chronic form ASTaspartate aminotransferase ALTalanine aminotransferase CB Conjugated bilirubin TBtotal bilirubin ALP alkaline phosphatase γGT gammaglutamiltransferase a increase of d decrease of absence of information due to the impossibility to compare continuous variables considering the short number of patients with alteration of this parameter Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 255 15 DIAGNOSIS 151 Mycological Diagnosis Diagnosis of PCM is established based on the demonstration of P brasiliensis in clinical samples 321 The fungus is visualised under plain optical microscopy whereby its morphology and reproduction by multiple exogenous budding typical of the parasitic form of the fungus allow for its identification Fig 1 However small forms might be confounded with Histoplasma capsulatum var capsulatum or nonencapsulated strains of Cryptococcus neoformans especially on histopathological examination In such cases culture of samples inoculation into susceptible animals or an immunofluorescence test with fluoresceinlabelled hyperimmune serum is needed The identification of P brasiliensis in sputum is more difficult than in skin lesion scraps and lymph node materials in which the amount of fungi is large Initially the technique merely involved direct observation of fresh samples on slides with covers lips Subsequently sputum clarification with 10 KOH Fig 1A or 4 NaOH and homogenisation were successively suggested 322 The rate of positive results of examinations using homogenised sputum samples is much higher compared to those using cleared sputum only The aforementioned techniques allow for the identification of fungus in sputum samples in the vast majority of cases According to current recommendations mycological testing should be performed in sputum samples collected over three consecutive days and a new sample should only be collected when initial testing is negative Another useful technique is the cell block Fig 1B preparation of sputum in paraffin followed by staining of sections with haematoxylineosin HE Fig 1C and methenamine silver GomoriGrocott 323 324 This technique allows for slides to be conserved for many years preserving of the paraffin blocks with included sputum and the preparation of new sections that can be stained for acidfast bacilli or neoplastic cells This technique is rather expensive and demands considerable time and is indicated when direct mycological examination yields negative results It should be noted that silver staining facilitates visualising the fungus which is a valuable help Fig 1D The sensitivity of the available methods for the investigation of P brasiliensis in sputum tends to be somewhat lower for patients with lung radiological lesions of the purely interstitial type for whom the number of samples tested should be higher An assessment of routine diagnostic methods at a university hospital over 34 years showed that the sensitivity of direct mycological examination for various different clinical samples was 75 and 63 for sputum the sensitivity of cell block preparation of sputum was 95 325 P brasiliensis should be cultured in one of the following culture media Mycosel BBL or Mycobiotic Agar Difco SABHI Difco Sabouraud agar oryeast extract agar Sputum samples should be digested with pancreatin or N acetylLcysteine and then is inoculated into plates or tubes in appropriate culture media at room temperature Transformation of the filamentous into the yeastlike phase characteristic of P brasiliensis is obtained by inoculating the fungus onto Kelley medium with haemoglobin at 3536ºC Mycological examination can also be performed using tissue fragments ground in a sterile gral and then placed on a slide under a covers lip or cut with a razor blade and inoculated into the culture medium 152 Histopathological Diagnosis Fragments of tissue collected through biopsy and stained using the HE and methenamine silver methods are used to establish the histopathological diagnosis of disease HE staining allows for assessing the hosts inflammatory response the organisation of granulomas and the presence of the typical Paracoccidioides sp yeastcells In turn methenamine silver stains the fungus wall thus revealing the presence of fungi with characteristic exogenous sporulation that gives rise to the socalled mickey mouse form strongly suggestive of P brasiliensis In addition the steering wheel form can be observed which is pathognomonic for this fungus species Methenamine silver staining does not allow for assessing the inflammatory response in tissues Histopathological examination has a sensitivity of 97 for the diagnosis of PCM 325 Histopathological examination allows both establishing a diagnosis of PCM and determining its severity according to the type of granulomas that are present compact in patients with preserved cellmediated immunity and loose among patients with severe depression of cellmediated immunity 256 The Open Microbiology Journal 2017 Volume 11 Mendes et al 153 Serological and Molecular Diagnosis 1531 Antibody Investigation General considerations Serological tests for the detection of antiP brasiliensis antibodies are helpful for diagnosis of PCM and represent indicators of severity as well as criteria for monitoring the response to specific treatment The diagnostic value of the various serological tests depends on their accuracy which is strictly related to the antigens used and the selected cutoff point Yeastlike cell culture filtrate is the antigen used in gel precipitation tests because it exhibits satisfactory diffusion in the gel Antigenantibody complexes precipitate due to their high molecular weight forming a macroscopically visible line In turn the polysaccharide antigen is used in complement fixation test CFT because it exhibits satisfactory fixation of the complement system which is the basis of this test Along the last decades we have observed the preparation of Paracoccidioides sp antigens in different Services in house without a standardized protocol leading to innumerable problems such as reproducibility and repetitiveness 326 As a consequence results obtained in different centers can hardly be compared In addition the identification of different species P brasiliensis and P lutzii and the P brasiliensis cryptic species has suggested the use of antigens prepared from the dominant species in the region the patients come from It is another difficulty for the appropriate interpretation of the serological tests 327 328 Possible crossreaction with antibodies in the serum of patients with other diseases fungal or not is a permanent source of concern This problem can be minimised by standardising the antigens used The identification of the P brasiliensis 43kDa glycoprotein gp43 represented a significant contribution to the knowledge of the immune response in PCM 19 Consequently any antigen formulation adequate for serodiagnosis must include this molecule 329 However some P brasiliensis isolates do not express gp43 330 P brasiliensis cultures are composed of various clones most of which do not initially secrete gp43 Only after 10 subcultures do these isolates begin to express the gene related with the secretion of this glycoprotein albeit in a variable manner 331 Aiming to improve the serological diagnosis of PCM much effort has been devoted to avoid the use of crude antigens and replacing them with recombinant proteins 332 333 which in many cases reduce the frequency of cross reactions 334 However the production of these molecules is expensive which limits their use at research centres 335 Antibody isotypes to a P brasiliensis somatic antigen were evaluated by ELISA in PCM patients with AF and CF who showed no differences in total IgG IgG1 IgG2 or IgG3 Higher levels of IgG4 were observed in AF patients while IgA predominated in CF patients However these isotypes have not been routinely assessed in the diagnosis and treatment followup of PCM patients Tests used The main tests developed for diagnosis of PCM are CFT agar gel precipitation tests DID and counter immunoelectrophoresis CIE indirect immunofluorescence IIF immunoenzymatic tests ELISA magnetic ELISA MELISA inhibition ELISA dotblotting and western blotting The sensitivity of CFT varies from 695 to 93 and its specificity ranges from 967 to 100 the predictive values being over 90 336 337 Table 4 However CFT stopped being used because it is difficult to perform requires large amounts of reagents 22 338 and is characterised by frequent crossreactions 339 Table 4 DID for diagnosis of PCM was introduced by Restrepo 23 the values of the accuracy parameters are high and the test results correlate with efficacious treatment 340 DID is the most widely used serological test for the diagnosis of PCM being considered as the standard by the authors of the Manual of Epidemiological Surveillance 340 CIE with antigens extracted from a sonicated cell suspension exhibits a sensitivity of 77 a specificity of 95 and an accuracy of 94 Table 4 341 Although simple to perform the latex agglutination LA slide test is scarcely used Its sensitivity is lower compared to the gel precipitation tests DID and CIE but has a negative predictive value of over 80 Table 4 The formulation with crude antigen should be preferred over the formulation with an ethanoltreated antigen because the former is easier to prepare and exhibits less frequent crossreaction with tuberculosis Table 4 the main differential diagnosis of PCM 342 One study assessed the LA test using a pool of crude fungal exoantigens and found a sensitivity of 84 and a specificity of 81 however a crossreaction with aspergillosis and histoplasmosis was observed Table 4 343 Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 257 IIF was also used for diagnosis of PCM 344 as advantages it can be used with anticomplementary sera 345 and allows for characterisation of the isotypes of antiP brasiliensis immunoglobulins 24 Despite its high sensitivity IIF exhibits frequent crossreactions especially with sera from patients with histoplasmosis 346 Table 4 The need for a fluorescence microscope subjective readings and manual steps are limitations of this method 347 ELISA affords several advantages Specifically the reagents last a long time when they are safely stored and the test is simple to perform provides quantitative results and has high sensitivity The test can detect antibodies in concentrations as low as 005μgml 345 However its sensitivity and specificity vary according to the antigen used and the selected cutoff point Using P brasiliensis yeastlike cell culture filtrate as the antigen and a titre of 180 as cutoff point the sensitivity and specificity of ELISA reached 100 348 However it exhibited crossreactions with histoplasmosis cryptococcosis aspergillosis and lacaziosis lobomycosis Table 4 In one study that employed cytoplasmic antigens 66 of the tested sera reacted at titres equal to or higher than 1128 and the specificity of the test was 95 However this test exhibited crossreaction with histoplasmosis 36 Table 4 349 The reaction of antibodies from PCM patients with gp43 predominantly involves peptide epitopes 85 350 The crossreaction of serum antibodies from patients with histoplasmosis or lacaziosis has been attributed to galactose containing carbohydrate epitopes in Nlinked carbohydrate chains of gp43 This occurs when methods using plastic immobilised antigen eg ELISA are performed ELISA is easy to perform and presents high sensitivity but low specificity Never the less the improvement of its specificity using sodium metaperiodate antigen gp43 treatment serum absorption process with Candida albicans or Histoplasma capsulatum antigens and dilution of serum galactose showed poor results 351 In spite of several immunological methods useful for PCM diagnosis many of them are timeconsuming lack accuracy are expensive result in crossreactions or are unavailable for routine clinical laboratories justifying the proposal of new tests In this regard dotblotting is a practical rapid and less expensive method Table 4 352 353 DotELISA method has been proposed to detect antiP brasiliensis antibodies in sera from patients with PCM using membranes impregnated with fungal antigens It has advantage to be used by laboratories with few resources or even directly in the field In addition it presents high values of the accuracy parameters 354 Table 4 Western blotting or immunoblotting have been employed to detect antiP brasiliensis antibodies in sera from patients with PCM using membranes impregnated with fungal exoantigens Using gp43 and gp70 the sensitivities for diagnosing PCM were 100 and 96 respectively However this test exhibited crossreactions with sera from patients with histoplasmosis Table 4 Although immunoblotting has high sensitivity this test has not been standardised as routine practice in most laboratories 355 Table 4 Parameters of accuracy of serological tests for diagnosis of paracoccidioidomycosis Detection of serum antibodies Cross reactions with other diseases Accuracy Parameters Method Antigen S E PPV NPV A PLR NLR Ref CF a FL 930 967 930 967 955 310 006 336 CF a FL 695 1000 1000 1000 849 345 030 337 RFC Po 339 DID FL 762 1000 1000 900 930 380 023 23 CIE FL 770 950 940 341 LA Crude Ethanol 696 609 800 830 615 622 851 822 767 760 345 352 037 047 342 LA Crude 840 1000 1000 710 880 210 015 343 IFA FL 980 820 970 950 940 1225 001 346 ELISA b FL 1000 1000 1000 1000 1000 1250 0014 348 ELISA c CT 660 950 940 680 780 132 035 349 ELISA gp43 1000 350 Dotblot p 27 recombinant 1000 1000 1000 1000 1000 111 009 352 Dotblot gp43 1000 353 DotELISA FL 910 954 960 982 930 354 258 The Open Microbiology Journal 2017 Volume 11 Mendes et al Accuracy Parameters Method Antigen S E PPV NPV A PLR NLR Ref IB gp43 gp70 1000 960 355 IDD CFAPl exoPl TCAPl 100 588 176 1000 1000 1000 1000 1000 1000 1000 895 220 356 ELISA d FL 960 969 950 951 950 950 960 970 955 960 192 3167 004 005 368 Cross reactions Method Antigen TBC HST ASP CRC LSZ CCD ESP Ref CF a FL 33 235 38 336 CF a FL 00 142 00 00 00 337 RFC Po 526 600 00 652 339 DID FL 00 00 00 23 CIE FL 341 LA Crude Ethanol 170 320 469 344 474 158 342 LA Crude 272 267 343 IFA FL 80 340 20 346 ELISA b FL 330 550 220 500 550 00 348 ELISA c CT 360 00 00 349 ELISA gp43 531 307 350 Dotblot p 27 recombinant 00 00 100 00 00 00 352 Dotblot gp43 00 00 00 313 353 DotELISA FL 354 IB gp43 gp70 1000 425 00 00 00 00 355 IDD CFAPl exoPl TCAPl 356 ELISA d FL 414 207 358 348 435 285 368 Evaluated in healthy donors Evaluated in patients with microbiologically confirmed active disease DID double agar gel immunodiffusion test ELISA enzymelinked immunosorbent assay CF complement fixation CIE counterimmunoelectrophoresis IFA indirect immunofluorescence assay LA latex agglutination test IB immunoblotting assay cutoff a 18 b 180 c 20 d 0710 and 0850 FL culture filtrate antigen yeast phase CT cytoplasmic antigen Po polysaccharide antigen p 27 recombinant proteinEthanol antigen treated with ethanol ExoPl crude exoantigen of P lutzii CFAPl cell free antigen of P lutzii TCAPl crude exoantigen of P lutzii precipitated with trichloroacetic acid S sensitivity E specificity PPV NPV positive and negative predictive values A accuracyPLR positive likelihood ratio NLR negative likelihood ratio TBC tuberculosis HST histoplasmosis ASP aspegillosis CRC cryptococcosis LSZ lacaziosis CCD coccidioidomycosis ESP esporotrichosis Diagnosis of P lutzii The investigation of antibodies in sera from patients with PCM likely caused by P lutzii was assessed using three methods and three types of antigens obtained from strain EPM 2008 356 The best results were achieved by the DID test and using cell free antigen CFA with sensitivity specificity positive and negative predictive values and accuracy of 100 1532 Antigen Investigation Antigens are investigated using known specific antibodies to detect antigenantibody reactions As an advantage there is no dependence on antibody production by the patient which is impaired in immunosuppressed cases In one study inhibition ELISA using BALBc mice monoclonal IgG antibodies targeting an 87kDa protein obtained from P brasiliensis yeast cell culture filtrate had a sensitivity of 80 and a specificity of 100 however the frequency of crossreaction with sera from patients with aspergillosis histoplasmosis cryptococcosis and sporotrichosis was high 357 Table 5 In another study an investigation was made of antigens in the serum bronchoalveolar lavage fluid and CSF from patients with PCM Using antigp43 and antigp70 monoclonal antibodies a sensitivity of 90 to 96 and a specificity of 96 to 100 were observed 358 Table 5 Despite the success of this method in both the diagnosis and followup of patients after the onset of treatment its viability for use in the clinical routine is unknown 359 Table 4 contd Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 259 In one study the detection of circulating P brasiliensis gp43 and gp70 in the CSF of patients with neuroPCM had a sensitivity of 909 and a specificity of 100 Table 5 360 In another study P brasiliensis antigens were detected in the urine of patients with active PCM before the onset of antifungal treatment with the 43 and 70kDa antigens being the most frequently observed antigens 361 Antigen investigation was also useful for the monitoring of treatment given that reactivity decreased in the samples collected during clinical recovery and increased in the case of relapse Table 5 Parameters of accuracy of serological tests for diagnosis of paracoccidioidomycosis Detection of serum antigens Cross reactions with other diseases Accuracy parameters Method Monoclonal antibodies S E PPV NPV A PLR NLR Ref InhiELISA a FL 800 1000 1000 690 860 200 02 357 InhiELISA b gp43 962 967 962 967 965 320 003 358 InhiELISA c gp43 gp70 909 1000 1000 967 975 300 009 360 Cross reactions Method Monoclonal antibodies TBC HST ASP CRC LSZ CCD ESP Ref InhiELISA a FL 555 400 1000 100 111 357 InhiELISA b gp43 00 00 358 InhiELISA c gp43 gp70 360 Evaluated in healthy donors Evaluated in patients with microbiologically confirmed active disease InhiELISA InhibitionELISA cutoff a 33µgmL b1353µgmL c 023gmL for gp43 and 097gmL for gp70 FL culture filtrate antigen yeast phase S sensitivity E specificity PPV NPV positive and negative predictive values A accuracy PLR positive likelihood ratio NLR negative likelihood ratio TBC tuberculosis HST histoplasmosis ASP aspegillosis CRC cryptococcosis LSZ lacaziosis CCD coccidioidomycosis ESP esporotrichosis Excellent article review was recentily published focusing the different methodologies for the identification of specific antibodies and antigens used in serological diagnosis of PCM 362 1533 Molecular Diagnosis Molecular methods allow for confirming the aetiology of disease through the identification of DNA fragments specific to P brasiliensis with no need to culture the fungus Among these methods polymerase chain reaction PCR and nested PCR stand out Nested PCR was employed to detect P brasiliensis DNA fragments through the use of primers derived from the gene that encodes gp43 and detection of a 196base pair bp sequence 363 The use of molecular markers is important for diagnosis of PCM as well as for ecological molecular and epidemiological studies of P brasiliensis in Latin America In this regard the 58S and 28S ribosomal genes and intergenic regions of P brasiliensis Pb01 were amplified and sequenced these products were able to distinguish P brasiliensis from other pathogenic fungi on PCR 364 In a clinical study that used specific primers designed from a 072kb fragment P brasiliensis DNA was detected in the sputum and CSF of patients with PCM 365 A PCR assay that employed primers derived from the gene that encodes gp43was able to detect P brasiliensis DNA in sputum the technique had a detection limit of 10 cellsmL of sputum 366 One study assessed sera from patients with suspected PCM by means of conventional PCR using primers for the ITS1 ribosomal DNA of P brasiliensis The results showed that this technique was not effective for detecting P brasiliensis DNA in serum 367 Although PCR and nested PCR are highly sensitive specific or rapid methods they are still not available for routine diagnosis of PCM especially in the countries where the disease occurs ie developing countries 1534 Diagnosis of Relapse Relapse is defined as the reappearance of PCM in patients who remained in a state of clinical radiological and serological cure for two years after discontinuation of antifungal treatment 368 260 The Open Microbiology Journal 2017 Volume 11 Mendes et al The DID test has low sensitivity for diagnosis of relapse 45 ELISA should therefore be used exhibiting a positive result in 80 of cases In addition the hypothesis of relapse cannot be ruled out without judicious mycological assessment 368 16 CASE DEFINITION PCM cases are defined as follows a confirmed cases characterised by the presence of suggestive clinical manifestations and detection of typical of the P brasiliensis yeast forms in clinical samples and b probable cases characterised by the presence of suggestive clinical manifestations and detection of specific antibodies in the serum by the DID test but without identification of P brasiliensis in clinical samples 177 c possible patient with at least one of the following clinical manifestation with a 4week duration after exclusion of tuberculosis and other diseases with similar symptomatology 1 cough with or without expectoration and dyspnoea 2 sialorrhea odynophagia hoarseness 3 any type of skin lesions 4 cervical or generalised lymphadenomegaly 5 child or young adult with hepatosplenomegaly andor abdominal mass 177 17 TREATMENT AND CONTROL OF CURE From its initial report by Adolfo Lutz in 1908 1 until the 1940s PCM was considered to be a fatal disease because of the lack of appropriate therapeutic agents Since the 1940s various drugs have been used to treat this disease with satisfactory results The first of these agents were sulfanilamides such as sulfapyridine 369 which were followed by the introduction of amphotericin B in 1958 which is a powerful broadspectrum antifungal agent Although this drug is efficacious in the treatment of PCM 370 it is quite difficult to manage because of its side effects and the need for long hospital stays for intravenous administration Then the efficacy of the trimethoprimsulfamethoxazole combination cotrimoxazole CMX was demonstrated and became widely used in the treatment of PCM 371 The development of imidazole derivatives at the end of the 1970s particularly ketoconazole KTC 372 373 further broadened the scope of therapeutic options Itraconazole ITC a triazolederivative was introduced at the end of the 1980s and proved to be 100 times more active in vitro against P brasiliensis than KTC 374 Regarding to its efficacy and safety ITC has become widely used 375 377 More recently voriconazole VRC a secondgeneration triazole derivative proved to be as effective as ITC in the treatment of PCM however this treatment has been reported to be less safe 378 Table 6 presents the drugs and dosages used to treat PCM Table 6 Antifungal compounds indicated in the treatment of paracoccidioidomycosis Antifungal compounds Route of administration Daily doses Intervals among doses hours Adiministration with food Concentration in CSF Amphotericin B deoxycollate IV increasing mdd 50 mg 48 Mínima Sulfadiazine PO 100 mg kg mdd 40 g 150mg kg ou 4g m 2 6 indifferent 40 a 60 SMXTMP IV PO 2400 mg 480 mg 810mg kg TMP 12 indifferent SMX40 TMP50 Ketoconazole PO 400 mg 58mg kg 24 no 2 h before or 2 h after 10 Itraconazole PO 200 mg 5mg kg 12 or 24 yes 1 Fluconazole IV PO 400 mg 12mg kg 12 or 24 indifferent 70 Voriconazole IV PO 400 mg 8mg kg 12 no 2 h before or 2 h after 50 SMXTMP trimethoprinsulfametoxazol combination TMP trimetoprima IV intravenous PO per os mdd maximum daily doses administration in alternate days increasing doses from 50 mg up to 10 mgkg every administration maximum dosis of 50mg Cerebrospinal fluid concentration porcentage of the plasmatic level 400 mg 1212h only in the first day pediatric dosage Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 261 18 CRITERIA OF CURE There are four criteria of cure of PCM clinical mycological radiological and immunological 379 181 Clinical Cure A patient is considered to have achieved clinical cure when the signs and symptoms of disease are no longer present and normalization of the erythrocyte sedimentation rate ESR The normalization of the ESR was included in the criteria of clinical cure to count with an objective parameter since the clinical evaluation can incorporate some degree of subjectivism 182 Mycological Cure Defined as negative results on mycological investigation for P brasiliensis occurring after efficacious treatment in the materials where it had been previously detected 183 Radiological Cure This criterion concerns the radiological assessment of the lungs as approximately 80 of patients exhibit the chronic form of PCM in which lung involvement is almost always present Radiological cure is attained when the radiological pattern becomes stable after treatment ie the same scar lesions are found on five radiographs taken every three months in the course of one year 184 Immunological Cure Immunological assessment includes evaluation of both humoral immunity through measurement of serum antiP brasiliensis antibodies and cellmediated immunity The serum levels of specific antibodies decrease after the onset of treatment to become undetectable on the DID test or stabilise at a very low concentration on the complement fixation test CFT This stabilisation is considered to be a serological scar Cellmediated immunity is seldom investigated after the onset of treatment 184 380 for which reason no test is suggested as routine 185 Apparent Cure This notion applies to patients with clinical mycological radiological and immunological cure for a twoyear period without receiving complementary treatment The term apparent cure should be preferred to cure to avoid the idea that a radical cure took place ie that the fungus was eradicated from the body Radical cure cannot be confirmed because foci with latent fungi certainly remain in the body after efficacious treatment 19 TREATMENT REGIMEN In the past PCM treatment was divided in two phases initial treatment in which amphotericin B was used until clinical cure was achieved andor cumulative dose limits were reached and complementary treatment in which sulphonamides derivatives were initiated and used until serological cure was achieved Currently both azoles and CMX may be used throughout the course of treatment However this treatment is also divided into two phases because the followup period differs from the initial treatment period During the initial treatment period patients are submitted to clinical serological haematological biochemical and radiological evaluations once a month until they achieve clinical cure and normal erythrocyte sedimentation rate During the complementary treatment patients are submitted to clinical serological and radiological evaluations every 3 months until one year has passed since serological cure was achieved Fig 9 381 In the last decade ITC and CMX were the main drugs used to treat PCM Then the Brazilian Guidelines for PCM 2006 indicated ITC as the first choice for treatment 177 However at this time only one unpublished study has compared these two treatments suggesting that ITC and CMX had similar efficacy however the duration of treatment was lower in patients who received the ITC 382 A study performed with 200 PCM patients to identify factors predicting cure showed that 864 of those treated with ITC were clinically cured while only 513 of those who received CMX achieved this outcome 382 However all the severe patients were treated with CMX a fact that may explain the difference in efficacy Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 263 The presence of associated diseases should be considered to prevent the occurrence of severe side effects For instance amphotericin B AmB should be avoided in patients with impaired kidney function and older adults with peripheral artery disease Azole derivatives in particular KTC and CMX are hepatotoxic and thus must be used cautiously in patients with liver disease As the incidence of alcoholism is high among patients with PCM the liver biochemistry should be monitored when these drugs are used In addition patients with other associated diseases also use other medications and thus the possibility of drug interactions should be considered For instance this is the case with patients receiving therapy for tuberculosis and KTC for PCM Rifampicin stimulates the degradation of KTC with consequent reduction of its serum concentration eventually to levels below those needed for antifungal activity In such cases the dose of KTC should be increased or the drug replaced by CMX or AmB Drugs that were previously inefficacious for PCM in a given patient must not be used in the same patient However it is important to bear in mind that PCM patients sometimes exhibit poor adherence to treatment or drop out thereby not representing true instances of drug resistance Sulphadiazine must be taken every six hours which makes compliance for appropriate treatment difficult often resulting in serum drug concentrations that are below those needed and consequent treatment failure 384 Although a rare occurrence PCM can affect pregnant or breast feeding women Azole derivatives are contraindicated under these circumstances while sulphonamide derivatives are contraindicated starting on the last month of pregnancy as they might cause kernicterus For this reason AmB is the first choice for treatment of pregnant women this drug is not teratogenic even though it crosses the placental barrier 385 21 SIDE EFFECTS Few studies assessed hepatobiliary variables after onset of treatment with ITC Some studies did not find any changes 376 378 while others did but without reporting their incidence or intensity 375 A recent study conducted with 200 patients found that the incidence of elevated hepatobiliary biochemistry after the onset of antifungal treatment with CMX or ITC was higher in AF compared to CF The pattern of abnormalities varied according to the antifungal compound used being hepatocellular for CMX 647 and mixedmild for ITC 600 The progression of these abnormalities also differed regarding to the antifungal compound used returning to normal with CMX but persisting among patients treated with ITC These abnormalities however did not demand require the discontinuation of the treatment in any case 318 Gastric discomfort occurred more frequently in the group treated with CMX compared to ITC 381 Transient elevation of the serum uric acid levels was more frequent among the patients treated with ITC 115 compared to CMX 33 The patients also exhibited transient reduction of the serum calcium ITC428 and CMX213 and phosphorus ITC233 and CMX25 levels 318 22 TREATMENT CONTROL In general clinical cure occurs in a relatively short time and gives patients the false impression that they are fully healed For this reason they should be made aware of the risk of recrudescence and of the consequent need for prolonged treatment and periodical assessment Fig 10 Mycological cure means that the fungus is no longer present only in the materials where it had been previously detected Appropriate methods applied by experienced mycologists are necessary for mycological cure to be safely established Mycological cure occurs even earlier the amount of fungi observed on direct examination decreases progressively until they are no longer found Healing of the mucosal and skin lesions and reduction of expectoration contribute to make P brasiliensis no longer present in samples The alveolar lesions disappear more rapidly compared to interstitial lesions which regress slowly Fig 6 Interstitial lesions exhibit variable behaviour the smaller nodules disappear with treatment while the larger ones persist even after the disappearance of the respiratory clinical manifestations and serum antiP brasiliensis antibodies become undetectable The most frequent residual disorders are pulmonary fibrosis and emphysema appearing as fibrotic streaks and nodules and diffuse or bullous emphysema 264 The Open Microbiology Journal 2017 Volume 11 Mendes et al Fig 10 Patient with the acutesubacute form of paracoccidioidomycosis A lymphnode enlargement of the suppurative type before treatmnent B the same patient after treatment with itraconazole The decrease of the antibody serum levels determined by agar gel precipitation tests observed in patients after treatment correlates with alterations of the secretion of cytokines increase of IL2 and IFNγ and reduction of IL10 Based on these findings the evaluation of the humoral immunity has been used as indicative of improvement of the cell mediated immunity 184 The lymphoproliferative response intradermal reaction to paracoccidioidin and balance between Th1 and Th2 cytokines return to normal after successful treatment 184 380 A study conducted with patients with the chronic form of PCM showed that the recovery of the cellmediated immunity as assessed through the quantification of mononuclear cell subpopulations and functional tests only occurred when the patients exhibited apparent cure 380 This correlation allows using apparent cure as a criterion of recovery of the specific cellmediated immunity responsible for keeping the surviving fungi in the latent state 23 IMMUNOSTIMULANTS Immunostimulants were first tested for treatment of PCM in an animal model with highly satisfactory results 386 387 However a single study assessed the progression of patients with PCM given antifungal agents and βglucan as an immunostimulantβglucan is β13polyglucose extracted from Saccharomyces cerevisiae and was used in a 10mg dose per intravenous or intramuscular routes once per week during the first month and then monthly for one year The patients treated with a combination of βglucan and an antifungal agent exhibited better progression in terms of clinical manifestations return to the normal erythrocyte sedimentation rate and humoral and cellmediated immunity compared to those not receiving immunostimulation 388 βglucan was found to behave as a powerful inducer of the production of tumour necrosis factor alpha TNFα and interferongamma IFNγ in BALBc mice these findings may account for the adjuvant effects of βglucan in the treatment of PCM 389 For this reason βglucan should be indicated for treatment of severe forms of disease provided the patients TNFα levels can be monitored as it is harmful to patients in excess 24 PROPHYLAXIS The lack of knowledge regarding the ecological niche of Paracoccidioides sp hinders the development of prophylactic measures that are likely to prevent infection among the population most exposed to the fungus 25 DISCUSSION Perhaps the only measure with some practical value for this population is the recommendation not to use plant leaves for anal hygiene While this practice does not prevent inoculation with the fungus which is highly improbable it Paracoccidioidomycosis Current Perspectives The Open Microbiology Journal 2017 Volume 11 265 seeks to avoid the fixation of fungi occasionally present in the bloodstream after having been inhaled The reason is that the sequelae derived from anal lesions may be very severe especially when they extend to the rectum CONCLUSION Investigators and laboratory technicians who work with Paracoccidioides sp samples should be very careful when handling culture media and infecting experimental animals In case of accidents likely to result in infection the involved area should be immediately washed with soap and water In addition the individual should be subjected to investigation for serum specific antibodies by means of DID and receive itraconazole 200mg one single daily dose after breakfast for one month If no clinical manifestations appear namely lesions on the probable site of inoculation attended by regional lymphadenopathy nor seroconversion occurs the medication should be discontinued and the patient subjected to clinical and serological assessment for an additional two months The absence of clinical manifestations and persistent negative serology allows discontinuance of monitoring Conversely the presence of PCM lesions or seroconversion indicates that antifungal treatment should be maintained and the patient should be assessed and monitored according to the instituted regimen CONSENT FOR PUBLICATION Not applicable CONFLICT OF INTEREST The authors declare no conflict of interest financial or otherwise ACKNOWLEDGEMENTS Declared none REFERENCES 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access article distributed under the terms of the Creative Commons Attribution 40 International Public License CCBY 40 a copy of which is available at httpscreativecommonsorglicensesby40legalcode This license permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited RESUMO DO ARTIGO PARACOCCIDIOIDOMICOSE PERSPECTIVAS ATUAIS DO BRASIL A paracoccidioidomicose PCM é uma doença granulomatosa sistêmica que pode acometer qualquer órgão do corpo predominantemente os pulmões órgãos ricos em células do sistema fagocitário mononuclear a mucosa do trato aerodigestivo superior UADT a pele e as glândulas adrenais Esta condição é causada por fungos termicamente dimórficos do complexo Paracoccidioides brasiliensis P brasiliensis P lutzii Pb01 e espécies Pb01like É uma doença endêmica limitada à América Latina do México à Argentina Os fungos do gênero são termicamente dimórficos podendo ser cultivados como células de micélio ou levedura Cultivada a 25ºC após 15 a 30 dias observase uma colônia branca tornandose aveludada e acastanhada Quando cultivados em meios sem carboidratos mas com substratos naturais os artroconídios aleuroconídeos e artroaleuroconídios apresentam 2 a 5µm de diâmetro A 37º C e em tecidos humanos e animais P brasiliensis se assemelha a células de levedura Seu crescimento é lento apresentando colônias rugosas e pregueadas de 7 a 20 dias após a inoculação A temperatura é o único fator desencadeante do dimorfismo do P brasiliensis quando o ÿ13glucano o principal polissacarídeo neutro da parede celular constituinte da fase patogênica da levedura substitui quase inteiramente o ÿ glucano que compreende o polissacarídeo neutro do micélio vegetativo Estágio Essa transição se comporta como um mecanismo de escape e a ÿ13glucana como um fator de virulência Ocorre que apesar dos importantes avanços no conhecimento da biologia dos agentes etiológicos da paracoccidioidomicose PCM estamos longe de ter um quadro completo de fatores biológicos relevantes como a ecologia desses agentes Poucos relatos estão presentes na literatura existente que abordam o isolamento desses patógenos do ambiente e embora o isolamento de fungos das fezes de morcegos e pinguins e alimentos de cães contaminados com solo tenha sido relatado apenas observações casuais com limitadas reprodutibilidade foram feitas Embora existam vários indícios de que P brasiliensis e P lutzii apresentem diferentes distribuições geográficas este assunto está longe de ser resolvido Enquanto isolados de P lutzii foram detectados principalmente nas regiões central e oeste do Brasil esta espécie já foi isolada de um paciente e detectada molecularmente em amostras de aerossóis da região sudeste Estudos experimentais sugerem que a capacidade de produzir conídios infectantes difere entre grupos genéticos ou espécies crípticas de Paracoccidioides o que por sua vez pode determinar a incidência de infecção Por exemplo os genótipos S1 e PS2 ocorrem mais comumente no Sudeste do Brasil a uma taxa de 91 e a produção de conídios foi identificada como sendo maior em S1 do que em isolados PS2 em condições de laboratório A respeito da infecção em animais domésticos e silvestres registrase que são maiores os estudos realizados em populações humanas Entretanto as a avaliações de infecções fúngicas em animais têm sido realizadas principalmente por meio de testes intradérmicos inquéritos sorológicos análises histopatológicas ferramentas moleculares e isolamento do patógeno em cultura A maioria desses estudos foi realizada no Sul e Sudeste do Brasil áreas de alta endemicidade de PCM Os resultados dos exames levaram à conclusão de que a comparação entre os animais revelou maior prevalência de infecção por P brasiliensis e P lutzii em cães e por ambas as espécies em animais silvestres Além disso a prevalência de soropositividade variou em relação à origem geográfica dos animais A descoberta de tatus de nove faixas Dasypus novemcinctus naturalmente infectados por P brasiliensis constituiu um marco no estudo ecológico desse patógeno levando a um melhor entendimento de sua possível distribuição na natureza Cabassous centralis tatu de cauda nua também foi encontrado naturalmente infectado na Colômbia como foi demonstrado pela primeira vez isolando este fungo de tecidos de tatu e confirmando sua identificação pelo sequenciamento das regiões ITS e GP43 Esses achados mostram que a infecção por P brasiliensis e P lutzii em animais de áreas endêmicas para a doença humana é mais comum do que se imaginava assim é importante que os veterinários estejam cientes do potencial de infecção por Paracoccidioides Sobre a epidemiologia a ocorrência de PCM tem sido relatada do México à Argentina sendo o maior número de pacientes de cinco países Brasil Venezuela Colômbia Equador e Argentina O Chile é o único país sulamericano sem nenhum caso autóctone relatado devido às condições climáticas desfavoráveis à sobrevivência do fungo no solo No Brasil as regiões Sudeste CentroOeste e Sul apresentam o maior número de casos A doença predomina no sexo masculino devido à proteção conferida pelo estrogênio que inibe ou dificulta a transformação de conídios e fragmentos de micélio na forma leveduriforme que é patogênica Os aspectos genéticos de tais fungos e as doenças infecciosas por ele causadas são complexos se relacionando também com o hospedeiro A constatação de que apenas 2 dos indivíduos expostos a espécies de Paracoccidioides desenvolvem PCM favorece a hipótese de que um componente genético desempenha um papel no equilíbrio entre infecção e doença Os dados sobre a suscetibilidade genética humana para PCM são escassos e nenhum resultado foi replicado ou validado Alguns genes candidatos já foram testados mas o pequeno número de indivíduos avaliados tem sido uma limitação para esses estudos Além disso a seleção de controles para estudos de associação genética é um ponto crítico e a exposição ao patógeno deve ser levada em consideração Assim os controles ideais para estudos de PCM são indivíduos que vivem e trabalham em áreas rurais de risco há muito tempo O teste intradérmico positivo realizado em indivíduos saudáveis com antígenos específicos deve ser utilizado como critério de seleção de controle pois este teste tem sido um bom marcador para confirmar uma exposição fúngica Uma informação importante sobre o tema é que o tabagismo que é muito comum entre pacientes com PCM aumenta o risco de PCM pulmonar em 10 vezes e reduz em 8 anos a idade dos pacientes no início dos sintomas A respeito da patogênese destacase que os pulmões são a porta usual de entrada do Paracoccidioides sp no corpo humano os esporos atingem os bronquíolos terminais e alvéolos causando áreas de pneumonite O epitélio respiratório representa o local primário no qual ocorre o contato entre os propágulos do fungo e os hospedeiros As células envolvidas na infecção humana pelo fungo incluem macrófagos alveolares e células epiteliais alveolares A partir dessas áreas o fungo se espalha pelos linfáticos para os linfonodos paratraqueais e parabrônquicos onde desencadeia uma reação granulomatosa As áreas com pneumonite constituem o polo parenquimatoso da infecção paracoccidioide e os linfonodos regionais acometidos constituem o pólo linfático Juntos o polo parenquimatoso linfangite ascendente e acometimento do linfonodo satélite é conhecido como complexo primário PCM Quando a resposta imune é insuficiente no momento da formação do complexo primário os fungos se multiplicam e se espalham primeiro pelo sistema linfático e depois pela via hematogênica para vários órgãos e sistemas Nesses casos a doença se manifesta imediatamente após a infecção ou seja após o primeiro contato com o fungo PCM doença pode progredir para morte ou cura A cura está associada à formação de cicatriz nos órgãos afetados podendo eventualmente causar sequelas dentre as quais se destacam a fibrose pulmonar e o enfisema com consequente comprometimento funcional As cicatrizes podem ser estéreis ou conter fungos viáveis que podem causar recidiva A capacidade dos patógenos de colonizar seus hospedeiros é altamente dependente dos mecanismos que permitem que o patógeno supere as barreiras físicas e imunológicas impostas pelo hospedeiro Para evitar a eliminação rápida os patógenos podem aderir rápida e efetivamente às células hospedeiras A capacidade do tipo de células de interagir umas com as outras de maneira ordenada depende de múltiplas interações adesivas entre as células e seu ambiente extracelular adjacente mediadas por moléculas de adesão celular que funcionam como receptores de superfície celular que podem desencadear sinais físicos e bioquímicos que regulam um grande número de funções como proliferação celular expressão gênica diferenciação apoptose e migração celular e são usados como porta de entrada para alguns patógenos Em razão da entrada primária na área pulmonar os achados patológicos estudados pelo artigo são focados no acometimento pulmonar Este é caracterizado por processos inflamatórios granulomatosos durante os quais a intensidade e o padrão morfológico dependem do estado imunológico do hospedeiro duração da infecção sem tratamento e virulência do fungo De acordo com o grau de imunidade os granulomas podem ser extensos com macrófagos linfócitos e plasmócitos dispersos Por outro lado pacientes imunocomprometidos podem desenvolver reações pneumônicas com alveolite difusa composta predominantemente por neutrófilos e afetando tanto a região hilar quanto a periférica dos pulmões Potenciais infecções por P brasiliensis são geralmente avaliadas com base em características histomorfológicas típicas através do uso de microscopia de luz em ambientes clínicos apropriados Um citoplasma multinucleado que é separado da parede celular com um espaço claro ou halo pode às vezes ser reconhecido em cortes corados por HE Para melhor diagnóstico da PCM a coloração com prata metenamina de Gomori GMS deve ser realizada para determinar os aspectos morfológicos desse fungo Em geral as lesões decorrentes da PCM são centradas nos eixos broncovasculares das vias aéreas proximais e 9 RESPOSTA IMUNE associada a achados radiológicos consistentes com padrão de asa de borboleta Em pacientes com doenças crônicas a que é reforçada pela resposta imune T helper 1 Th1 polarizada Os linfócitos Th1 efetores são recrutados para processo inflamatório granulomatoso promove ativação miofibroblástica e aumento da deposição de matriz extracelular O início a progressão e os resultados clínicos da PCM são influenciados por fatores ambientais pelo sistema imunológico do hospedeiro o local da infecção e liberar IFNy que aumenta a ativação de macrófagos Consequentemente o parênquima e a remodelação vascular pulmonar resultam em melhorias na fibrose radial tração A orquestração desses mecanismos pode levar ao aparecimento e progressão da doença baseiase em três grandes princípios 1 A PCM é uma doença endêmica e acomete indivíduos saudáveis ou seja aqueles sem A duração da sintomatologia da PCM é curta em pacientes com FA variando de algumas semanas a alguns bronquiectasias e distorção da arquitetura pulmonar O resultado final é uma fibrose pulmonar às vezes acompanhada de cor pulmonale e morte O envolvimento pulmonar é raro em pacientes jovens no entanto pode ocorrer em 5 a 11 dos casos Por esta razão o diagnóstico é confirmado apenas na autópsia Assim em áreas endêmicas essa possibilidade deve ser considerada sempre que um paciente apresentar antecedentes epidemiológicos para PCM ou a evolução clínica não for satisfatória após o uso de terapia antimicrobiana para doenças pulmonares comuns Em pacientes com micose sistêmica como a PCM a presença de lesões cutâneas e mucosas são iniciadores de diagnósticos específicos Em conjunto as características dessas lesões incluindo seu número morfologia clínica e acessibilidade para biópsia e coleta de material para exame direto e cultura fornecem indicadores valiosos que podem possibilitar o diagnóstico precoce As lesões cutâneas geralmente ocorrem como consequência da disseminação fúngica hematogênica geralmente originandose dos pulmões ou evoluindo de forma contígua a partir de lesões mucosas e muitas vezes apresentandose com envolvimento labial O envolvimento dos linfonodos submandibulares foi relatado pela primeira vez por Lutz O linfotropismo do P brasiliensis foi sugerido por Haberfeld 1919 e Niño 1939 apontou para uma relação direta entre mau prognóstico e o aparecimento precoce e gravidade da linfadenopatia A relevância do envolvimento linfonodal pode ser avaliada com base em sua frequência em estudos clínicos e de autópsia na detecção de envolvimento subclínico nas alterações do sistema linfático identificadas por avaliações linfográficas e cintilográficas e mais particularmente na depressão da resposta imune mediada por células resultantes de danos no tecido linfóide O envolvimento da mucosa do trato aerodigestivo superior UADT ou seja fossa nasal cavidade oral orofaringe hipofaringe e laringe é de grande relevância devido à sua alta frequência e à facilidade de coleta de amostras para identificação do agente etiológico Rouquidão dor e dificuldade em engolir ardor na garganta sensação de abaulamento ou ferida na boca e dispneia são as manifestações clínicas mais comuns As lesões na mucosa podem ser muito dolorosas principalmente quando se come alimentos quentes ou muito salgados Em geral mais de uma área do TSD está acometida sendo a maioria das lesões na laringe seguida da orofaringe hipofaringe e cavidade oral que apresentam prevalências semelhantes A bilateralidade é quase sempre observada para todos os sítios mas a morfologia das lesões é muito variável O envolvimento do trato gastrointestinal foi relatado nos primeiros estudos de pacientes com PCM Um dos casos relatados por Lutz apresentava diarreia crônica cuja etiologia não foi estabelecida Viana descreveu um caso de PCM disseminada e a autópsia revelou ulcerações micóticas no íleo apêndice e cólon A introdução de novas técnicas para a investigação do trato gastrointestinal levou a mais relatos de novos casos embora os estudos com grandes séries de casos permaneçam escassos e os estudos que avaliam a extensão total do trato gastrointestinal sejam ainda mais raros As queixas gastrointestinais são relatadas por mais de 50 dos pacientes quando a interrogação visa o sistema digestivo Nesses casos sialorreia disfagia halitose dor abdominal sensação de inchaço pirose e anormalidades na motilidade intestinal são achados frequentes seguidos de regurgitação vômitos soluços e presença de massa abdominal O conhecimento do envolvimento de ossos e articulações na PCM foi obtido em relatos de casos ou pequenas séries de casos poucos estudos prospectivos e sistemáticos foram conduzidos Tais estudos relataram uma frequência de acometimento entre 16 e 20 Uma revisão de estudos sobre envolvimento ósseo publicados até 1964 também está disponível O acometimento medular geralmente ocorre em pacientes com a forma agudasubaguda da PCM sendo raro na forma crônica A biópsia da medula óssea fornece as melhores amostras para demonstrar a interação fungohospedeiro Já a ocorrência de convulsões em um paciente com lesões cutâneas disseminadas da PCM foi a primeira pista que aponta para um possível envolvimento do SNC A publicação subsequente de vários relatos de casos mostrou que esse distúrbio é mais comum do que se pensava anteriormente Os casos de PCM são definidos da seguinte forma a casos confirmados caracterizados pela presença de quadro clínico sugestivo manifestações e detecção de formas típicas da levedura P brasiliensis em amostras clínicas e b casos prováveis caracterizada pela presença de manifestações clínicas sugestivas e detecção de anticorpos específicos no soro por o teste DID mas sem identificação de P brasiliensis em amostras clínicas c possível paciente com pelo menos um da seguinte manifestação clínica com duração de 4 semanas após exclusão de tuberculose e outras doenças com sintomatologia semelhante 1 tosse com ou sem expectoração e dispneia 2 sialorréia odinofagia rouquidão 3 qualquer tipo de lesão de pele 4 linfadenomegalia cervical ou generalizada 5 criança ou jovem adulto com hepatoesplenomegalia eou massa abdominal Os critérios de cura dessa doença são divididos em quatro clínico micológico radiológico e imunológico Considerase que o paciente obteve cura clínica quando os sinais e sintomas da doença não estão mais presentes e a normalização da velocidade de hemossedimentação VHS A normalização da VHS foi incluída nos critérios de cura clínica por contar com um parâmetro objetivo uma vez que a avaliação clínica pode incorporar algum grau de subjetivismo Em geral a cura clínica ocorre em um tempo relativamente curto e dá aos pacientes a falsa impressão de que estão totalmente curados Por isso devem ser alertados para o risco de recrudescência e consequente necessidade de tratamento prolongado e avaliação periódica Definido como resultado negativo na investigação micológica para P brasiliensis após tratamento eficaz em 185 A cura micológica significa que o fungo não está mais presente apenas nos materiais onde havia sido detectado anteriormente Métodos apropriados aplicados por micologistas experientes são necessários para que a cura micológica seja estabelecida com segurança A cura micológica ocorre ainda mais cedo a quantidade de fungos observada ao exame direto diminui progressivamente até que não sejam mais encontrados A cicatrização das lesões da mucosa e da pele e a redução da expectoração contribuem para que o P brasiliensis não esteja mais presente nas amostras Já a cura radiológica diz respeito à avaliação radiológica dos pulmões pois aproximadamente 80 dos pacientes apresentam a forma crônica da PCM na qual o acometimento pulmonar está quase sempre presente A cura radiológica é alcançada quando o padrão radiológico tornase estável após o tratamento ou seja as mesmas lesões cicatriciais são encontradas em cinco radiografias realizadas a cada três meses ao longo de um ano A avaliação imunológica inclui a avaliação tanto da imunidade humoral através da dosagem sérica de antiP brasiliensis e imunidade mediada por células Essa noção se aplica a pacientes com cura clínica micológica radiológica e imunológica por um período de dois anos sem receber tratamento complementar O termo cura aparente deve ser preferido a cura para evitar a ideia de que ocorreu uma cura radical ou seja que o fungo foi erradicado do corpo A cura radical não pode ser confirmada porque focos com fungos latentes certamente permanecem no corpo após tratamento eficaz Para escolher qual remédio será o melhor aplicado há fatores a ser considerados Os casos graves devem ser tratados com a droga mais eficaz preferencialmente por via intravenosa pelo menos no início do tratamento para garantir alta biodisponibilidade do medicamento Medicamentos administrados por via oral devem ser evitados em pacientes com linfadenopatia abdominal mesmo quando nenhuma síndrome de má absorção é evidente Poucos estudos avaliaram as variáveis hepatobiliares após o início do tratamento com ITC Alguns estudos não encontraram alterações enquanto outros o fizeram mas sem relatar sua incidência ou intensidade RESUMO DO TEXTO HISTOPLASMA CAPSULATUM INFECÇÃO PULMONAR E IMUNIDADE A infecção por Histoplasma capsulatum causa morbidade e mortalidade significativas em todo o mundo Este fungo é um ascomiceto dimórfico que cresce em forma de hifa no solo e guano de pássaros e morcegos Após a inalação dos esporos o H capsulatum se transforma na fase de levedura patogênica Essa forma se replica dentro de macrófagos que transportam a levedura dos pulmões para praticamente qualquer órgão A indução da imunidade adaptativa particularmente a resposta Th1 é necessária para a ativação dos macrófagos e eliminação eficiente da levedura A exposição ao H capsulatum geralmente resulta em depuração assintomática no entanto a histoplasmose pode se manifestar como uma doença pulmonar aguda semelhante à gripe uma doença pulmonar cavitária crônica ou uma doença pulmonar progressiva Casos de histoplasmose ocorrem em todo o mundo mas estão concentrados em regiões endêmicas A área norteamericana está centrada nos vales dos rios Ohio e Mississippi Grandes pesquisas de testes cutâneos na década de 1960 revelaram que 80 dos homens adultos jovens desta região eram positivos para exposição ao H capsulatum A carga da doença é regional uma análise dos registros hospitalares dos EUA estimou 3370 internações e 254 mortes associadas à histoplasmose em 2002 com quase 90 das hospitalizações ocorrendo nos estados do centrooeste e do sul As Américas Central e do Sul contêm grandes áreas endêmicas e as revisões chamaram a atenção para a carga subdiagnosticada da histoplasmose associada ao HIV nessas regiões Focos endêmicos adicionais estão na China sudeste da Ásia subcontinente indiano Austrália e África Não se trata de um patógeno contagioso e se adapta especialmente em hospedeiros mamíferos Muitas espécies de mamíferos estão infectadas com H capsulatum e são hospedeiros acidentais que devem lidar com a capacidade de sobrevivência da levedura dentro dos macrófagos Na infecção humana e de camundongo os macrófagos fornecem um nicho para a proliferação de H capsulatum que não pode ser interrompido sem imunidade adaptativa Após o reconhecimento de H capsulatum é necessária uma mobilização celular apropriada para uma resposta imune eficaz Neutrófilos e macrófagos são recrutados precocemente para o local da infecção Os neutrófilos humanos são fungistáticos não fungicidas contra H capsulatum Após ativação celular via citocinas Th1 IFNÿ e GMCSF os macrófagos inibem o crescimento intracelular de H capsulatum Os mecanismos implantados por macrófagos murinos e humanos são distintos Enquanto a acidificação do fagossomo é usada por macrófagos murinos é dispensável em macrófagos humanos A fagocitose do H capsulatum estimula uma explosão respiratória dentro de macrófagos humanos mas não murinos No entanto estudos utilizando camundongos deficientes em NADPH oxidase sugerem que a inibição da replicação fúngica in vivo pode exigir a produção de ROS Mecanismos adicionais de inibição sugeridos por estudos in vitro incluem espécies reativas de nitrogênio e privação de metal Mesmo com ativação os macrófagos não podem esterilizar eficientemente os tecidos e assim abrigar o H capsulatum Macrófagos infectados induzem a formação de granulomas em pacientes imunocompetentes Embora os granulomas fossem considerados uma forma de proteção do hospedeiro evidências recentes sugerem que os granulomas induzidos por Mycobacterium tuberculosis servem como um repositório para organismos patogênicos A histoplasmose de reativação é uma entidade clínica reconhecida particularmente em indivíduos que deixaram áreas endêmicas em regiões que não são conhecidas por conterem o fungo Embora estudos na década de 1950 indicassem que os granulomas curados continham apenas organismos mortos a eficácia da recuperação do organismo é desconhecida Assim a fonte de organismos que causam a reativação permanece enigmática Como as células apresentadoras de antígenos mais potentes as DCs fornecem uma ligação entre a imunidade inata e adaptativa DCs humanas são capazes de matar H capsulatum Esta atividade fungicida foi dependente de hidrolases lisossômicas mas não da explosão respiratória ou produção de óxido nítrico As DCs humanas conduzem a proliferação de células T CD4 ou CD8 apresentando o antígeno de H capsulatum diretamente ou de macrófagos apoptóticos respectivamente Tratase de uma resposta imune adaptiva A privação de metais traço é uma estratégia imunológica eficaz para retardar ou interromper uma infecção Na histoplasmose murina a ativação de macrófagos envolve mecanismos que limitam pelo menos dois minerais essenciais ferro e zinco H capsulatum deve obter ferro ligado à ferritina ou transferrina no compartimento fagossomalisossoma Possui vários meios para conseguir isso incluindo sideróforos redutases férricas e manutenção de pH favorável Macrófagos murinos ativados com IFNÿ e lipopolissacarídeos neutralizam a aquisição de ferro por H capsulatum possivelmente via produção de NO inversamente o aumento da disponibilidade de ferro aumenta a sobrevivência da levedura Este mecanismo de controle de H capsulatum não foi relatado em macrófagos humanos e a importância do ferro na histoplasmose clínica não foi esclarecida Nossa compreensão da importância das células CD4 na histoplasmose surgiu a partir de observações em camundongos e humanos Em 1971 os médicos notaram que os pacientes com histoplasmose geralmente exibiam um aumento nos linfócitos T ou B enquanto os pacientes com doença disseminada progressiva tendiam a não ter essa resposta Estudos iniciais em camundongos atímicos confirmaram a importância das células T para o controle da Histoplasmose Na década de 1980 a epidemia de HIV trouxe um aumento dramático na incidência gravidade e conscientização da histoplasmose Uma resposta CD4 prejudicada é o principal fator de risco a histoplasmose disseminada progressiva tipicamente se apresenta naqueles com contagens baixas de células T CD4 Os pacientes que tomam medicamentos que suprimem o número ou a função das células CD4 são outra grande população com risco aumentado para histoplasmose particularmente doença disseminada