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 Pb01-like 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.

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 yeast-like 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 Gilchrist’s 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 Lutz’s 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 Gonzalez-Ochoa suggested that the lungs are actually the entry point [6], a hypothesis that was reinforced by Mackinnon’s 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 cell-mediated and humoral immunity was established at the School of Medicine of Botucatu [16].

P. brasiliensis exhibits a complex antigenic structure that includes glycoproteins, glycopeptides, lipids and polysaccharides. The correlation between virulence and presence of α-1,3-glucan 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 43-kDa 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 Fava-Netto 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 follow-up 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 yeast-like phase, which confirmed Lutz’s original observation (mycelial phase in vitro and yeast-like phase in guinea pigs) was demonstrated by Negroni [25] and was introduced into the laboratory routine for the identification of P. brasiliensis. Fluorescein-linked immunoglobulin conjugates were also added to the techniques used for the identification of P. brasiliensis in clinical samples [26].

The depression of the cell-mediated 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 antigen-dependent [30].

In PCM, the various possible outcomes of the host-parasite 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 PCM-causing 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 long-lasting 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 (SVS-MS) [36]. This program was developed as an initiative of some states of the federation [36].

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 mother-cells present 20 to 30µm in diameter and can produce 10 to 12 daughter-cells, with 2 to 10µm, forming the characteristic pilot wheel. The mother-cell with two daughter-cells 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 – cell-block preparation of sputum (GMS, x400) showing the mickey-mouse 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). [B,C,D 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 hematoxilin-eosin (HE), which hardly stain the fungal wall. On the other hand, HE stain permits the identification of the preserved fungal cells.

The high prevalence of negative serological tests in mycologically confirmed patients from the Midwest Region using antigens from isolates in the Southwest Region (B-339) 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 Pb01-like [40]. The study of these and other isolates led to the description of a new species of Paracoccidioides, initially coined Pb-01, 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.

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 α-1,3-glucan, the major cell wall neutral polysaccharide constituent of the pathogenic yeast phase replaces almost entirely the β-glucan, that comprises the neutral polysaccharide of the vegetative mycelial phase. This transition behaves as a mechanism of escape and α-1,3-glucan like a virulence factor.

Despite important advances in knowledge of the biology of the etiological agent(s) 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 nine-banded 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 and/or 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 P.brasiliensis and P.lutzi 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 9:1 (S1:PS2), 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 acute/subacute 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 rainfall/humidity. 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 2-20cm 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 well-documented cluster of cases of the acute/subacute form of PCM, with potential relationship with the El Niño Southern Oscillation (ENSO), phase in 1982-1983 [66]. The ENSO behaviour explains the rainfall variability not only in the Brazilian equatorial region, but also in the Southern. The atypically high soil water storage in 1982-1983 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.

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 (weeping-capuchins 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 post-inoculation; 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 road-killed 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 nine-banded 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 (naked-tailed 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 co-evolved in concert [60]. Of the 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 two-toed 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.

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 host’s immune response to infection with Paracoccidioides sp determines the progression of the host-parasite 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, i.e., the so-called 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, Moscardi-Bacchi M, Rezkallah-Iwasso M, Montenegro MR. Paracoccidioidomycosis. Baillière’s Clin Trop Med Commun Dis. 1989; 4.1: 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, i.e., 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.

The yeast phase is the pathogenic form of the P. brasiliensis. High concentrations of α-1,3-glucan and low concentrations of galactomannan in the cell wall of the yeast-like forms are correlated with P. brasiliensis virulence [104, 105].

In a murine model, gp43 antigenaemia led to depression of the cell-mediated 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 acute/subacute form of disease [107]. These findings suggest that gp43 might have a considerable immunomodulating effect, maintaining the depression of the cell-mediated 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.

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 host-fungus 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 host-Paracoccidioides 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 IL-6 and IL-8 levels in Paracoccidioides sp/A549 cell cultures. Therefore, these interactions have been found to be associated with increases in the host’s 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 and facilitating the invasion of the host [130-134]. Additionally, the 14-3-3 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, pre-treatment 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 Paracoccidioides-host interaction is the Src family (SFKs), and the host-fungus interaction process may involve the activation these kinases and extracellular signal-regulated kinase 1/2 (ERK1/2) 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 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 activation, and the secretion of IL-8 and IL-6 that is promoted by this fungus have been reported to be dependent upon activation of p38 MAPK and ERK 1/2 in A549 cells [143].

The ability of these pathogens to induce apoptosis may be an important virulence factor since it reduces the host's 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 caspase-2, 3 and 8 [147]. Moreover, Paracoccidioides sp may modulate epithelial cell (A549) apoptosis via the expression of apoptotic molecules, such as Bcl-2, Bak and caspase-3, 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 14-3-3 and gp43 adhesins had substantial influences on this process. Additionally, apoptosis may be mediated by Fas-FasL, and CTLA-4 was identified to be involved in modulating immune responses in patients infected with PCM [151].

To further increase the understanding of Paracoccidioides-host interactions, the use of mammalian cells models may facilitate the establishment of increased knowledge related to how fungus-host 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].

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 immune-competent patients reveals multiple well-formed granulomas composed of cohesive clusters of histiocytes and organized centrally with peripheral admixed lymphocytes; however, some eosinophils and/or 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 H&E-stained sections. To better diagnosis PCM, Gomori’s methenamine silver staining (GMS) should be performed to determine the morphological aspects of this fungus. Through the use of GMS and other staining methods, such as Papanicolaou and periodic acid-Schiff (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].

The involvement of the mucous membrane of the upper aerodigestive tract - UADT, i.e., 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].

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 Pupo’s 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, mulberry-like 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 laryngeal-ventricle 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 patient’s 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 <7.0), 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.

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 cell-mediated 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 17-hydroxycorticosteroid 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 semi-synthetic 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 Addison’s disease [251], suggesting that the measurement of ACTH might be useful for early diagnosis of chronic adrenal 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].

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 acute/subacute 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 sub-obstruction, and consequently into acute abdomen that requires surgical treatment.

Some patients exhibit protein-losing 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 protein-losing enteropathy. Lymphatic stasis, due to abdominal lymphadenopathy, might cause hypertension of the lymphatic system, with consequent extravasation of the protein- and lymphocyte-rich 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 serum albumin in the absence of renal loss or impaired synthesis. Neither clinical nor radiological assessment is able to demonstrate the occurrence of protein-losing enteropathy. For this reason, the faecal excretion of ⁵¹Cr-labelled 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 D-xylose 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 acute/subacute 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 tomography-guided puncture aspiration of the pancreatic head contained pancreatic epithelial cells and several P. brasiliensis yeast-like cells [266].

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 pre-existing 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 lung-involved 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 one-third of cases with bone involvement [267].

Skeletal scintigraphy with 99mTc-methylene diphosphonate (MDP) is a highly valuable technique for the detection of PCM lesions due to its high sensitivity, the early appearance of abnormalities, assessment of the full skeleton in a single exam, rare contraindications, non-invasiveness 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.

Bone marrow involvement usually occurs in patients with the acute/subacute form of PCM, being rare in cases with the chronic form. Bone marrow biopsy provides the best samples to demonstrate the host-fungus 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].

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 on-going 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 (7/34) or meningitic (3/34) [279]; lesions were found in the brain (13/24), cerebellum (6/24) or both (5/24) [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 ofγ-globulin, 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 ring-enhancing 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 T₂-weighted scans.

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 and/or the presence of epidemiological antecedents denoting high risk of infection with P. brasiliensis in patients with neurological complaints

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 patient’s 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.

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 yeast-like 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 thyrotropin-releasing 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, i.e., primary, secondary or tertiary [293].

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].

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.

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 cell-mediated immune response and extensive dissemination of disease; it was for this population of patients that the term “mixed forms” was first suggested [298].

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].

(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 Addison’s 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 hard-to-treat sequelae, which occasionally require surgical intervention. Lesions in the gastrointestinal tract might cause obstruction or sub-obstruction, requiring two-stage surgical correction. Skin and mucosal lesions are very often disfiguring, especially laryngeal lesions, causing considerable and often irreversible impairment of the patient’s voice. Other organs may also develop sequelae.

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 4-5% of patients with PCM have comorbidity HIV infection [307-309], and approximately 1.5% 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 PCM-HIV regularly used cotrimoxazole prophylaxis for the prevention of PJP.

Additionally, the epidemiological factors associated with fungi exposure could explain the low observed prevalence of Paracoccidioides/HIV co-infection. 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 HIV-infected individuals using the gp43 intradermal test, we found that 12.2% of HIV patients were co-infected 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 co-infection prevalence of 47% previously identified among rural settlers [91].

Patients with the PCM-HIV 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 ≤ 200cells/mm³. 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 HIV-positive than HIV-negative patients [307]. Involvement of the mononuclear phagocyte system is characteristic of acute/subacute 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 antigen-based 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 co-infection has previously been observed in 9.4 to 58.3% of cases with PCM-HIV comorbidity [307, 310]. Secondary prophylaxis with antifungal agents until T CD4⁺ lymphocytes levels reach at least 200cells/mm³ has been suggested based on studies assessing other opportunistic mycoses [177].

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 and/or their treatments could reactivate quiescent P. brasiliensis foci. Although the co-morbidity PCM-lymphoma 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.

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].

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].

The changes most frequently found in both AF and CF are reductionsin serum cholesterol and albumin levels and elevated levels of α₁-acid 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 γ-globulin, mucoproteins and α-1-acid glycoprotein were previously reported [320].

AF – acute /subacute form; CF- chronic form; AST-aspartate aminotransferase; ALT-alanine aminotransferase; CB- Conjugated bilirubin; TB-total bilirubin; ALP- alkaline phosphatase; γ-GT – gamma-glutamiltransferase; [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.

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 non-encapsulated strains of Cryptococcus neoformans, especially on histopathological examination. In such cases, culture of samples, inoculation into susceptible animals or an immunofluorescence test with fluorescein-labelled 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 haematoxylin-eosin (HE) Fig. (1C) and methenamine silver (Gomori-Grocott) [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 acid-fast 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-acetyl-L-cysteine and then is inoculated into plates or tubes in appropriate culture media at room temperature. Transformation of the filamentous into the yeast-like phase, characteristic of P. brasiliensis, is obtained by inoculating the fungus onto Kelley medium with haemoglobin at 35-36ºC.

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.

Fragments of tissue collected through biopsy and stained using the H&E and methenamine silver methods are used to establish the histopathological diagnosis of disease. H&E staining allows for assessing the host’s 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 so-called “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 cell-mediated immunity and loose among patients with severe depression of cell-mediated immunity.

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.

Defined as negative results on mycological investigation for P. brasiliensis occurring after efficacious treatment in the materials where it had been previously detected.

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, i.e., the same scar lesions are found on five radiographs taken every three months in the course of one year.

Immunological assessment includes evaluation of both humoral immunity, through measurement of serum anti-P. brasiliensis antibodies, and cell-mediated 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.

Cell-mediated immunity is seldom investigated after the onset of treatment [184, 380], for which reason no test is suggested as routine.

This notion applies to patients with clinical, mycological, radiological and immunological cure for a two-year period without receiving complementary treatment. The term apparent cure should be preferred to cure to avoid the idea that a radical cure took place, i.e., 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.