Paracoccidioidomycosis

Description of the Pathogen

Until 2006 the genus Paracoccidioides was thought to consist of a single species, P. brasiliensis, considered the sole etiologic agent of paracoccidioidomycosis. At present however, molecular and genetic studies have revealed that this genus includes a complex of five distinct phylogenetic species (S1a, S1b, PS2, PS3, PS4) that appear to be confined to distinct regions within the endemic zones. Recently, the P. brasiliensis spp. complex has been formally renamed, with the proposed names P. brasiliensis for S1, P. americana for PS2, P. restrepiensis for PS3, and P. venezueliensis for PS4. Additional studies suggest that yeast morphology slightly, but significantly, differs across all five Paracoccidioides spp., an observation that may indicate that these phylogenetic species could well be designated as formal taxonomic species. In addition, genomic comparison has indicated that P. brasiliensis is related to the uncultivable pathogen Lacazia loboi .

Of note, a highly divergent monophyletic species, Paracoccidioides lutzii, has been described. Distinguishing P. lutzii is based on a distinctive genome and differences in the conidia, as well as on morphology and size of the daughter yeast cells. So far, P. lutzii has only been identified in central, southwestern, and northwestern Brazil. Species within the complex P. brasiliensis also appear confined to particular endemic regions.

Species within the Paracoccidioides genus exhibit the phenomenon called dimorphism, implicating that they can give rise to colonies corresponding to a mold or to a yeast, with temperature being the only recognized factor known to date that is capable of triggering this change. The latter phenomenon takes place when the β-glucan predominating in the mold is replaced almost entirely by the yeast-phase cell wall polysaccharide α-1,3-glucan.

As for the Paracoccidioides genus classification, the relevant species are assigned to the family Ajellomycetaceae, order Onygenales. Genomic analyses support the existence of a sexual cycle in species of the Paracoccidioides genus, by detection of heterothallic groups (mating type 1-1 [MAT1-1] or MAT1-2), with gene expression in certain isolates but with no production of fruiting sexual bodies.

Since 2009 the Broad Institute at the Massachusetts Institute of Technology (Cambridge, MA) has completed the whole-genome sequencing database for the P. brasiliensis spp. complex, which is publicly available in the Sequence Read Archive database ( https://www.ncbi.nlm.nih.gov/sra ).

In cultures incubated at 35°C to 37°C the fungus grows in about 10 days, producing a soft, convoluted, tan-to-cream–colored colony, microscopically composed of yeast cells of varying size (4–40 µm), some adopting the characteristic multiple-budding formation (pilot's wheel). Isolated, round-to-oval yeast cells and short chains of blastoconidia are also observed, as well as large broken yeasts ( Figs. 267.1 and 267.2 ). Viable yeasts have refractive cell walls and prominent lipid vacuoles.

P. brasiliensis has been repeatedly recovered from human clinical samples, tissues of the nine-banded armadillo (Dasypus novemcinctus) and, more rarely, from the northern naked-tailed armadillo (Cabassous centralis). On occasion, P. brasiliensis has also been isolated from dogs and detected molecularly in other animal species.

As indicated earlier, this fungus encompasses two morphotypes: a mold at temperatures less than 28°C and a yeast in mammalian tissues and cultures at 35°C to 37°C. Both morphotypes require ample oxygen supply for their growth. At temperatures less than 28°C, including 4°C if in liquid substrates, the fungus grows within 3 weeks and produces white, cotton-like colonies having short tufts of aerial mycelia. Microscopically, only chlamydoconidia and thin septated hyphae are observed in media such as Sabouraud, but in carbohydrate-reduced media, the fungus may produce conidia (size <5 µm), especially if grown in sandy and claylike soils rich in water. Conidia respond to temperature changes by germinating into hyphae at 20°C to 24°C or converting into yeasts, both in vitro (36°C) and in infected mice; in the latter, conidia initiate a progressive pulmonary process ending in fibrosis and extrapulmonary dissemination.

P. brasiliensis undergoes an intricate thermal transition process by switching from a mycelial saprobic form, probably in nature, to a virulent yeast form in infected mammal tissues. This transition involves expression of an array of virulence factors (cell glucan type, proteolytic enzymes, melanins, fungal adherence factors, and others). In addition, biofilm formation has been demonstrated and shown to correlate with increased expression of adhesins and hydrolytic and other types of enzymes. All of the given factors facilitate P. brasiliensis interactions with its accidental mammalian host controlled by certain genes expressed during host adaptation.

Ecology and Epidemiology

Paracoccidioidomycosis, a noncontagious disease, is peculiarly restricted to Latin America, from Mexico (23° N) to Argentina (34° S), with Brazil accounting for the largest number of patients (>80%) and originating mainly from the States of São Paulo, Paraná, Rio Grande do Sul, Goiás, Rio de Janeiro, and Rondônia. Venezuela, Colombia, Ecuador, Peru, Bolivia, and Argentina follow in rank with significantly fewer cases. Uruguay, Paraguay, and the Central American countries (Nicaragua, Belize) report few or no cases. The mycosis is extremely rare in the Caribbean Islands, with only single cases having been reported from Trinidad, Grenada, and Guadalupe. The mycosis is unknown in Chile, Surinam, and Guyana. In addition, the mycosis appears to prefer a humid environment and surrounding woods.

The main accidental hosts of Paracoccidioides spp. are men and armadillos, especially the northern nine-banded Dasypus novemcinctus, although other animals have also been implicated, presumably infected in rural or periurban environments. As a rule, the primary infection is asymptomatic or gives rise to nonspecific symptoms that rarely progress to disease manifestations. No epidemic outbreaks or transmission between individuals have been reported. Due to lack of knowledge on the precise habitats of the fungus, suspected to occur in rural settings, and based on experimental animal infections, it is accepted that the fungal infecting structures, the conidia (<5 µM), are inhaled and reach the lungs, where the infection is usually controlled by the immune cellular responses but may leave behind scarred focal points containing latent yeast cells that may reactivate years later, giving rise to full-blown disease.

The disease is characterized by long periods of latency—30 or more years—as demonstrated by the nonautochthonous cases reported outside recognized endemic areas (North America, Europe, Asia). In every case, however, the patient had previously resided in an endemic country, where the primary infection should have occurred, as exemplified by cases reported in Spain, France, Portugal, and Japan, among others.

Areas of endemicity center around tropical and subtropical forests, where mild temperatures (17°C–24°C), high annual precipitations (2000–2999 mm), and abundant waterways are common, conditions that favor certain agricultural crops (e.g., coffee, tobacco). Changes in traditional agricultural practices were implicated in the increased number of juvenile cases reported in Rio de Janeiro, Brazil. Two large Brazilian studies emphasized the connection between agriculture-related jobs and paracoccidioidomycosis, pointing toward closer or repeated contacts with P. brasiliensis in soil. Nonetheless, the fungus has been isolated only sporadically from the environment, a fact that, together with the prolonged mycosis latency periods and absence of outbreaks, has contributed to maintaining the uncertainty about the P. brasiliensis microniche. In an attempt at defining more precisely the fungus’ environmental niche, Barrozo and coworkers studied the role of weather conditions prevailing at the time of diagnosis of a number of juvenile patients, those with the shortest disease course. Absolute air humidity, soil, water storage, and presence of the Southern Oscillation Index influenced significantly the incidence, thereby suggesting their connection with acquisition of the infection. Imprecise knowledge of the fungal habitat has also hindered definition of the infection route, although clinical and experimental animal studies have ruled out traumatic implantation and pointed instead toward the aerosol route as the portal of entry.

The incidence of paracoccidioidomycosis is difficult to estimate due to the noncompulsory report of cases and, in addition, to the influence of climate changes and also by human migration and occupation of large unexplored territories where medical care would not be available, as indicated by Martinez. Nonetheless, the incidence of this mycosis was estimated for Brazil, the country with the largest number of patients—10 to 30 cases per 100,000 inhabitants per year. However, variations were recorded, as indicated in the Itaipu Lake region, where 102 patients were diagnosed in a period of 18 months, and in the State of Rondônia they had the closest to a “prolonged” outbreak, as 39.1 cases per 100,000 inhabitants were reported from 1997 to 2012.

The age and gender distribution of clinical cases is unusual. The mycosis is rare in children (<2%) and teenagers (<9%), with the remaining patients being 30 years of age and older. Several different series encompassing more than 5000 patients revealed that the male-to-female ratio was 11 : 1. This proportion contrasts with the infection rate as determined by a reactive paracoccidioidin skin test, which had a similar prevalence for both genders. Of note, when the disease occurs in prepubertal patients, no major gender difference becomes apparent, as found with more than 100 children in whom the boy : girl ratio varied from 1.2:1.3 to 1.1:1.0.

The occupational distribution reveals a predilection for individuals who have been engaged in agriculture (>60%) or have had soil-related jobs with subsequent exposure to infected soil and dust, mainly in connection with coffee, cotton, tobacco, and sugar cane plantations. Increased acreage of sugar cane fields in the southwestern region of Brazil may alter these data because this crop requires pesticide application and plant burning, a combination that may affect Paracoccidioides spp. saprobiotic life in nature. Other occupations mentioned as relatively frequent are masonry and lumberjacking.

The presence of Paracoccidioides spp. in soils or other types of habitats in Latin America, South America in particular, indicate that a large territory is to be taken as endemic and, furthermore, considering that the most prevalent occupation for the working population of such territory would be agriculture-related, including crops manipulation, P. brasiliensis airborne infection could represent a problem for newcomers. According to Martinez the existence of unexplored territories, such as those along the Amazon River running through various South American countries, would attract migrants pursuing occupation of promising uninhabited land open to agriculture-related endeavors. This means that a naïve population from these diverse countries may well become exposed to P. brasiliensis, leading to infection, which may result in continued expansion of the endemic area, as already observed in north and center-west Brazil. Nonetheless, progression of the infection to the disease stage and the clinical form that may become manifested in infected people would depend on various host factors: genetic background, demography, living conditions, presence of immunosuppression, and others. New endemic areas usually have medical care deficiencies, which would cause considerable social impact. Even if the corresponding clinical manifestations would not become apparent in a short period, P. brasiliensis latency poses a challenge, mainly considering that the fungus may become active with time, giving rise to a chronic disease that would cause suffering, disability to the patient, and leave sequelae even in the presence of specific treatment.

Deaths due to paracoccidioidomycosis resulted from extensively disseminated lesions, respiratory and adrenal gland insufficiency, and several other complications, mainly those attributed to residual fibrosis. In Brazil the death toll was estimated as 3.48 per million inhabitants, thus making paracoccidioidomycosis the fifth most common cause of death among the chronic infectious diseases of the lower respiratory tract. During the period 1998 – 2006, a Brazilian study was performed covering 35% of the country and representing 27% of the 5560 Brazilian municipalities, including the larger part of the endemic paracoccidioidomycosis area, consisting of an infected population estimated at 4.3 per 1 million inhabitants. The results analyzed 6732 hospitalization events attributable to paracoccidioidomycosis (82% male). The corresponding results estimated a global mortality of 1.4 per one million inhabitants, with 60% of such deaths attributed to the mycosis itself and the remaining to the fibrotic sequelae. In two case series from different endemic Brazilian areas, lethality reached 6.1% and 7.6%, respectively. Nonetheless, at the end of treatment, when assessing patients who did not require hospitalization, lethality was zero. Considering a 5% mortality rate as a mean value, it is possible to estimate the number of new annual cases of this mycosis in Brazil as 3360.

Brazilian authors studying paracoccidioidomycosis, and assessing the number of new patients, observed the large group of cases with a prolonged follow-up and the medical-social characteristics of the mycosis as mentioned earlier, and this revealed the wide dimension of this mycosis as a public health problem in Brazil and emphasized this condition as a neglected disease.

Pathogenesis and the Immune Response

After the initial contact with Paracoccidioides spp., a subclinical infection may develop, which, if the host is unable to control it, becomes clinically apparent. There are two main clinical presentations—the acute/subacute, or juvenile, and the chronic, or adult disease _. The former is characterized by marked involvement of the reticuloendothelial system and occurs in less than 15% of cases _, whereas the chronic or adult type of disease involves the lungs and characteristically results in extrapulmonary lesions. This is the predominant form occurring in approximately 90% of cases and probably represents endogenous reactivation after years of initial contact with the fungus. In addition, a residual form is also recognized and is represented by fibrotic scarring occurring at the sites of previously active lesions. A recent cluster analysis done in patients with concomitant lung, mucosal, and skin lesions has strongly supported the existence of two different sets of patients. The first analysis includes patients with mucosal damage, odynophagia, and/or dysphagia, plus alveolointerstitial infiltrates, and the second one consists of patients exhibiting dermal lesions, dyspnea, and lung fibrosis. The former conditions would represent early stages of the infection, whereas the latter would correspond to a more chronic granulomatous and fibrosing process.

Paracoccidioides spp. infection may become dormant but may be reactivated later under the influence of ill-defined conditions prevalent in rural settings, such as chronic alcoholism, malnutrition, and smoking.

The pathogenesis of this mycosis depends partly on Paracoccidioides spp. virulence factors and their antigenic composition, on environmental conditions, and also on the efficiency of the host immune response. Different in vitro, in vivo, in silico, and ex vivo experiments, as well as clinical studies, have demonstrated that specific and nonspecific defense mechanisms, both innate and adaptive, are crucial in building host resistance. In consequence, this response is highly complex and multifactorial, but when a particular balance of the host-fungus interaction is achieved, the infection is controlled and progression to overt disease is halted; otherwise, individuals who have lost this balance develop clinically manifested paracoccidioidomycosis. Thus the initial interaction of the microorganism with the first line of host defenses would determine the type of immune response to be developed (protective, deleterious), and, accordingly, would determine the disease outcome.

Paracoccidioides spp. have several virulence factors. As an example, the fungus cell wall components—glycoproteins and polysaccharides—control the fungus morphologic transition from the conidia or mycelial morphotypes (26°C) to the parasitic yeast (37°C). This form switch is obligatory if the pathogen is to adapt and thrive inside the host in whom the infection is established. Furthermore, on its surface P. brasiliensis expresses several adhesins, such as the glycoprotein gp43 plus the 19-kilodalton (kDa), 30-kDa, and 32-kDa proteins, all fulfilling similar functions, as well as a series of enzymes (malate synthase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, enolase). The adhesins allow the fungus to recognize the host extracellular matrix protein molecules (laminin, fibronectin, fibrinogen, plasminogen) that facilitate not only fungal adherence but also invasion and dissemination to other organs and systems. This fungal pathogen also produces melanin and exhibits the capability to form biofilms in vitro and in vivo, factors that confer resistance to both a host's microbicidal mechanisms and to antifungal therapy.

Clinical Manifestations

Paracoccidioidomycosis has been divided into three categories—one lacking clinical manifestations and another two directly related to the disease processes and largely dependent on age and host immune status.

• 1

  Paracoccidioidomycosis infection

  This is considered to be a latent, clinically asymptomatic process lasting 1 month to years after the initial fungal contact and that may later evolve to progressive, clinically manifested disease. This form is acquired when a healthy individual enters in contact with Paracoccidioides spp. The infection stage is diagnosed by a reactive intradermal test to specific antigens and also at necropsy by demonstrating the presence of fungal cells in tissues.

• 2

  Paracoccidioidomycosis disease

  • A

    Acute/subacute disease (moderate or severe): This is an overt process evidenced by involvement of multiple organs with lymph node, liver, and spleen hypertrophy and also by manifestation of skin lesions, the latter often multiple and widely distributed throughout the body. The lungs do not often reveal radiographic abnormalities, nor are clinical manifestations obvious, despite the fact that P. brasiliensis is frequently detected in respiratory specimens. This form is usually diagnosed in children, young adults, and acquired immunodeficiency syndrome (AIDS) patients, as well as in those with other immune alterations.

  • B

    Chronic progressive disease/adult form (mild, moderate, or severe): This is the most common (90%) of the clinical presentations, diagnosed in older patients and characterized by important lung involvement and by frequent lesions in the mucosae, skin, adrenal glands, and other sites. Severe cases are defined by meeting three or more of the following criteria: (1) weight loss greater than 10% of the normal body weight; (2) intense pulmonary involvement; (3) involvement of other organs, such as adrenal glands, central nervous system (CNS), and bones; (4) presence of pseudotumoral (>2 cm diameter) lymph nodes in multiple chains, superficially or deeply located, with or without suppuration; and (5) high antibody titers.

    Mild cases are those with weight loss less than 5% and involvement of a few organs, with no dysfunction. In both clinically manifested forms, constitutional symptoms (fever, asthenia, general malaise, weight loss) are regularly observed.

• 3

  Residual form

  This is characterized by the presence of sequelae originating in the previously infected fibrous tissues, mainly in the lung but also reported in adrenal glands and mucosae.

Characteristics of the Lesions

Lungs

Lungs are the primary site of infection, but the corresponding clinical manifestations may be scarce; nonetheless, progression follows over time, and at diagnosis, symptoms (cough, expectoration, blood-tinged sputum, chest pain) and some degree of dyspnea are regularly noticed. Auscultation reveals minimal abnormalities compared with radiographic findings, with a clear dissociation between symptoms and imaging studies. The initial respiratory symptoms decrease or disappear fully after the onset of treatment, but dyspnea on heavy exertion might become worse and progress even with moderate or mild exertion. Often noticed are pulmonary function alterations and abnormalities in ventilation-perfusion. Most (85%) patients exhibit the obstructive pattern that may be mild, moderate, or severe with no relation with the degree of involvement. Hypoxemia is a sequel occurring in approximately one-third of the cases. These findings, however, are difficult to interpret because the coexistence with smoking habits, resulting in an obstructive pattern involving the small airways, usually accompanied by an increase in the dead space and in the alveolar-arterial oxygen gradient.

Chest radiograph images reveal mostly interstitial infiltrates (64%), followed by mixed lesions consisting of nodular and alveolar infiltrates that are occasionally confluent, frequently bilateral, symmetrical, and located preferentially in the central and lower fields ( Fig. 267.3 ). High-resolution computed tomography demonstrates abnormal findings in greater than 90% of patients with chronic pulmonary disease, with the commonest abnormalities being interlobular septal thickening (88%), peribronchovascular interstitial thickening (78%), centrilobular opacities (63%), intralobular lines (59%), ground-glass opacities (34%), cavities (17%), and airspace consolidation (12%), with the “reversed halo sign” being observed in 10% of the cases. ( Fig. 267.3 ). Gallium-67 scans reveal pulmonary lesions in practically all patients, irrespective of the clinical forms, thus confirming the lungs as the main target. At diagnosis, one-third of the patients with long-lasting disease have serious pulmonary sequelae, such as fibrosis (32%) and bullae (27%), and indirect signs of pulmonary hypertension with right ventricle enlargement and even cor pulmonale.

A Brazilian study examined 1059 deaths caused by the mycosis and found that mortality could be attributed to pulmonary fibrosis, chronic lower airway diseases, and pneumonic processes, thus signaling the importance of the lung in paracoccidioidomycosis. Awareness of the given abnormalities emphasizes early diagnosis and prompt initiation of specific treatment, thereby reducing associated morbidity and mortality. Pleural involvement is detected in only 2% of cases on plain chest radiographs and is characterized by small effusions (<300 cc) and pleural thickening without effusion as observed in 60% of the autopsy cases.