Sporothrix schenckii and Sporotrichosis
Mônica Bastos de Lima Barros,1,*Rodrigo de Almeida Paes,2 and Armando Oliveira Schubach2
1National School of Public Health
2Evandro Chagas Clinical Research Institute, Fiocruz, Rio de Janeiro, Brazil
*Corresponding author. Mailing address: Fundação Oswaldo Cruz/Fiocruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro CEP 22045-900, Brazil. Phone: 55 21 2598 2518. Fax: 55 21 3415 2086. E-mail: rb.zurcoif@sorrablbm.
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Summary: Sporotrichosis, which is caused by the dimorphic fungus Sporothrix schenckii, is currently distributed throughout the world, especially in tropical and subtropical zones. Infection generally occurs by traumatic inoculation of soil, plants, and organic matter contaminated with the fungus. Certain leisure and occupational activities, such as floriculture, agriculture, mining, and wood exploitation, are traditionally associated with the mycosis. Zoonotic transmission has been described in isolated cases or in small outbreaks. Since the end of the 1990s there has been an epidemic of sporotrichosis associated with transmission by cats in Rio de Janeiro, Brazil. More than 2,000 human cases and 3,000 animal cases have been reported. In humans, the lesions are usually restricted to the skin, subcutaneous cellular tissue, and adjacent lymphatic vessels. In cats, the disease can evolve with severe clinical manifestations and frequent systemic involvement. The gold standard for sporotrichosis diagnosis is culture. However, serological, histopathological, and molecular approaches have been recently adopted as auxiliary tools for the diagnosis of this mycotic infection. The first-choice treatment for both humans and cats is itraconazole.
Sporotrichosis, caused by the dimorphic fungus Sporothrix schenckii, is currently distributed throughout the world, especially in tropical and subtropical zones. Infection generally occurs by traumatic inoculation of soil, plants, and organic matter contaminated with the fungus. Certain leisure and occupational activities, such as floriculture, agriculture, mining, and wood exploitation, are traditionally associated with the mycosis. Zoonotic transmission has been described in isolated cases or in small outbreaks. At present, veterinarians, technicians, caretakers, and owners of cats with sporotrichosis are regarded as a new risk category for the acquisition of the disease. The lesions are usually restricted to the skin, subcutaneous cellular tissue, and adjacent lymphatic vessels. Eventually, this fungus can disseminate to other organs, and alternatively, on rare occasions, inhalation of conidia may lead to a systemic disease. Several factors, such as inoculum load, immune status of the host, virulence of the inoculated strain, and depth of traumatic inoculation, influence the different clinical forms of sporotrichosis. The gold standard for sporotrichosis detection is culture; however, serological, histopathological, and molecular approaches have been recently adopted as auxiliary tools for the diagnosis of this mycotic infection.
Sporothrix schenckii was isolated for the first time in 1896 by Benjamin Schenck, a medical student at the Johns Hopkins Hospital in Baltimore, MD, from a 36-year-old male patient presenting lesions on the right hand and arm. This isolate, from the patient abscess, was then studied by the mycologist Erwin Smith, who concluded that the fungus belonged to the genus Sporotrichum (217). Previously, Linck in 1809 and Lutz in 1889 referred to some possible sporotrichosis cases, but the isolation of the fungus by these authors for case definitions was not possible (126). The second undeniable sporotrichosis case was described in 1900 by Hektoen and Perkins, also in the United States (Chicago, IL). This was the case of a boy who suffered an injury with a hammer hitting his finger, with the lesion presenting spontaneous regression. These investigators gave the sporotrichosis agent its current denomination, Sporothrix schenckii (95). Later, this fungus was erroneously included in the genus Sporotrichum, which comprises basidiomycetous fungi which are neither dimorphic nor pathogenic for humans or other animals (216). This erroneous nomenclature remained until 1962, when Carmichael recognized differences in the conidiations of members of the genus Sporotrichum and isolates from sporotrichosis cases (37).
In 1903, Sabouraud suggested to Beurmann and Gougerot the use of potassium iodine for the treatment of sporotrichosis, which was a common disease in France during the beginning of the 20th century (126). This has hitherto been a satisfactory therapy for sporotrichosis, although no randomized, double-blind, placebo-controlled trials have ever been conducted (267).
The first reported case of natural animal infection was described in 1907 by Lutz and Splendore in rats from Brazil (141). The possibility of human infection by bites from these rats was considered (186). Also in Brazil, in 1908, Splendore reported the detection of asteroid bodies around Sporothrix yeast cells, which offer a very useful tool for sporotrichosis diagnosis in histological examinations (126, 196).
Sporothrix schenckii belongs to the kingdom Fungi and is a eukaryotic organism that is without mobility and heterotrophic and presents chitin on its cell wall. For several years, this fungus was included in division Eumycota, subdivision Deuteromycotina, class Hyphomycetes, order Moniliales, and family Moniliaceae (128). After a substantial fungal taxonomy revision by Guarro and coworkers, this fungus was characterized in division Ascomycota, class Pyrenomycetes, order Ophiostomatales, and family Ophiostomataceae (84).
The sexual form of S. schenckii is as yet unknown. However, there is substantial molecular evidence that this fungus undergoes recombination in nature (163). Nevertheless, some studies imply that S. schenckii in an ascomycete, since it presents a simple septum, with Woronin bodies (237) and three chitin synthase genes (44).
Molecular analyses of the 18S region of the ribosomal DNA indicate that the sexual form of S. schenckii could be Ophiostoma stenoceras (22). On the other hand, morphological and physiological studies exhibit consistent differences between these two species. O. stenoceras is unable to produce dematiaceous conidia, as does S. schenckii. Also, S. schenckii does not produce perithecium on malt, rice, or potato media, as is observed for isolates of O. stenoceras (60, 181). Differences are also apparent when these species are inoculated in mice. S. schenckii can be found in several tissues from all infected mice after intravenous inoculation, and O. stenoceras is detected in certain organs from some infected animals (59). These observations lead to the conclusion that the O. stenoceras anamorph and S. schenckii are different species. Meanwhile, other molecular studies (56, 97), together with work by Berbee and Taylor (22), reinforce that the S. schenckii teleomorph is classified in the genus Ophiostoma. Berbee and Taylor highlight that S. schenckii belongs to the pyrenomycete lineage, lacking forcible ascospore discharge (22).
Recently, Marimon and coworkers (150), on the basis of phenotypic and genotypic analyses, suggested that S. schenckii should not be considered the only species that causes sporotrichosis, and based on macroscopic characteristics, sucrose and raffinose assimilation, ability to grow at 37°C, and the nuclear calmodulin gene sequence, they described four new species in the Sporothrix complex: (i) S. globosa, a fungus distributed worldwide (145, 180); (ii) S. brasiliensis, the species related to the zoonotic epidemic of sporotrichosis in Rio de Janeiro, Brazil (150, 179); (iii) S. mexicana, limited to Mexico (150); and (iv) S. luriei, formerly S. schenckii var. luriei (151), differing from S. schenckii mainly in the tissue form by the production of large, often septate budding cells unable to assimilate creatinine or creatine (53). On the other hand, other authors support its separation by rRNA internal transcribed spacer (ITS) sequence data (54). Another species, S. cyanescens has been isolated from blood and skin samples from human patients, but pathogenicity studies conclude that, although this fungus can grow at 37°C, it is avirulent (233). Figure 1 presents a key to differentiate species within the S. schenckii complex (151).
Identification key for Sporothrix species of clinical interest, based on morphological and phenotypic tests described by Marimon and collaborators (152). PDA, potato dextrose agar; CMA, corn meal agar.
Recently, de Meyer and collaborators (56) described three other environmental Sporothrix species, S. stylites, S. humicola, and S. lignivora. The first two species differ from S. schenckii by the inability to produce melanized conidia and the consequent nondarkening of colonies with age. S. lignivora has distinctive conidia that do not match in size and shape those of other Sporothrix or Ophiostoma species. It is interesting to note that isolates classified as S. humicola were previously referred to as environmental isolates of S. schenckii. In their study, the authors concluded that β-tubulin sequence analysis is strongly recommended for taxonomic studies of Sporothrix species isolated from the environment.
SPOROTHRIX CELL BIOLOGY
Sporothrix schenckii is a dimorphic fungus. In its saprophytic stage or when cultured at 25°C, it assumes a filamentous form, composed of hyaline, septate hyphae 1 to 2 μm wide, with conidiogenous cells arising from undifferentiated hyphae forming conidia in groups on small, clustered denticles. These one-celled conidia are tear shaped to clavate (53) and do not yield chains (233). Often, hyaline or brown thick-walled conidia arise beside the hyphae. The dark cell walls of the conidia distinguish S. schenckii from other, nonpathogenic Sporothrix species (56, 242). Macroscopically, filamentous colonies in media such as malt extract agar or potato dextrose agar (Fig. 2 ) are often smooth and wrinkled, white to creamy at first and then turning brown to black after a few days (126, 170, 196). Some strains, however, have the ability to form dark colonies from the beginning of growth (5). The S. schenckii colonies never become cottony or floccose (126).
Cultures of pus from lesions of S. schenckii-infected patients. Most strains become visible after 4 days of growth on Sabouraud dextrose agar, presenting no visible dark pigment at this stage (tube at left), whereas others are melanized since the beginning...
This fungus is evident in both human and animal tissues as budding yeasts. Yeastlike cells can be observed in various sizes and shapes. They may be round to oval, with 2- to 6-μm diameters, and usually have elongated, cigar-shaped buds on a narrow base. Macroscopically, yeast colonies (Fig. 2) are smooth, tan, or cream colored (130). Some molecular aspects implicated in proliferation and maintenance of this morphological form of S. schenckii involve calcium/calmodulin-dependent protein kinases (260) and a signaling pathway involving the interaction between a cytosolic phospholipase and protein G. Studies have proven that this pathway is necessary for the reentry of S. schenckii yeast cells into the budding cycle, suggesting its function in the control of dimorphism in this fungus and for the maintenance of the yeast form (259).
The transition from mold to yeast form in S. schenckii can be attained by culturing mycelia or conidia on rich culture media such as brain heart infusion agar at 35 to 37°C (170). Some strains, especially those related to the S. globosa species, may require lower conversion temperatures, since they do not grow well at 37°C (150). Although rich media are required for the mycelium-to-yeast transition, S. schenckii yeast cells can be maintained at 37°C in other media, such as Sabouraud dextrose agar. This transition process also occurs after patients are infected with filamentous S. schenckii. Morphological transformation at the ultrastructural level occurs by direct formation of budlike structures at the tips and along the hyphae together with oidial cell formation after septation of the hyphae, without conspicuous alterations of the cytoplasmic content of parent mycelial cell. There is no direct budding of yeast from conidiospores (77).
The beginning of the yeast-to-mycelium transition in S. schenckii is a process regulated by calcium, which induces both RNA and protein synthesis on the yeast cell (199). A prerequisite for this transition process is a nuclear division; afterwards, a germ tube is originated from the parental yeast cell and a septum is formed at mother cell-germ tube formation (25). It is interesting to note that yeast cells can be maintained at 25°C if cultured in liquid media with glucose and with the pH around 7.2 (195, 260).
Like other fungi, S. schenckii has a cell wall surrounding the plasma membrane in both the mycelial and yeast forms. There are characteristic differences in cell wall thickness between conidia, yeast forms, and filaments, as well variations in plasma membrane invaginations among these three morphological forms of the fungus. It has been shown by freeze fracturing studies that in conidia invaginations are short and abundant and in yeast forms they are scarce and longer, while the plasma membrane of the S. schenckii hyphae is smooth, without invaginations (244).
The fungal cell wall is rigid as well as complex, and recently it has been shown that S. schenckii produces vesicles that are probably related to the transfer of periplasmic molecules and pigment-like structures from the plasma membrane to the extracellular space, since in contrast to the case for prokaryotic organisms, in eukaryotic cells there is vesicular traffic of molecules to the plasma membrane (3, 197).
The chemical structures of fungal cell wall polysaccharides and glycoproteins have been studied basically because of the knowledge of the antigenic structures of human pathogens (193). A peptide-rhamnomannan was isolated from the yeast S. schenckii cell wall, where there were the polysaccharides d-mannose (50%) and l-rhamnose (33%), small amounts of galactose (1%), and about 16% peptides (138). Comparative studies of mycelial and yeast S. schenckii cell walls showed little difference in the glycosidic components. The cell wall composition of the mycelial phase included large amounts of lipids and protein and a lower concentration of mannose (193). The cell wall composition in S. schenckii conidial cells can also be affected by the time of culture, with a decrease in the rhamnose molar ratio and an increase in the mannose molar ratio (67).
The yeast cell wall of S. schenckii also contain granules of melanin (250) and proteins involved in adherence (135, 206), which contribute to fungal virulence. Of particular interest is a glycoprotein of 70 kDa isolated from the cell wall of the S. schenckii yeast phase. The purified glycopeptide has a pI of 4.1, and about 5.7% of its molecular mass is composed of N-linked glycans, with no evidence for O-linked oligosaccharides in this molecule. This glycoprotein has a uniform distribution on the fungal cell surface and participates in adhesion to the dermal extracellular matrix (206).
S. SCHENCKII GENETIC MATERIAL
Little is known about the S. schenckii genomic composition because this fungus is not amenable to genetic analysis based on meiotic segregation (256). Studies on the genomic DNA base composition rendered an average guanine and cytosine content of about 54.7 mol%, with the DNA showing a low degree of hybridization with O. stenoceras DNA, supporting the supposition that this fungus does not represent the sexual state of S. schenckii. However, 75% hybridization was observed with Ophiostoma minus DNA (159). More recently, Tateishi and coworkers (249), karyotyping eight strains isolated from patients in Japan, concluded that S. schenckii possesses six to eight chromosomes of 460 to 6,200 kb, with a total genome size of approximately 28 Mbp. Another study with strains from a different geographical origin predicted a 45-Mbp genome size for S. schenckii (256). Perhaps these differences are related to either the different species recently described (150) or to underestimations in the methods adopted for genome size determination. Also, it has been reported that S. schenckii is a diploid organism, bearing around 50 fg DNA per cell, in both the filamentous and yeast phases. On the other hand, aneuploidy, a state in which most of the chromosomes are disomic, cannot be excluded (256). It is interesting to note that diploidy is essential for thermal dimorphism in Cryptococcus neoformans, and similarities in life cycle between this fungus and other dimorphic fungi, including S. schenckii, may occur (232).
Studies on identification, typing, and epidemiology of sporotrichosis are usually based on mitochondrial DNA (mtDNA) analysis of restriction length polymorphisms (RFLP) with the restriction enzyme HaeIII. Initially, 24 mtDNA types were cited (136), and more recently types 25 to 30 (168) and 31 to 32 (103) were introduced. These analyses have been adopted in several studies with S. schenckii strains from different geographical origins and also environmental isolates (13, 103, 104, 243, 266).
S. SCHENCKII PHYSIOLOGY
Even though sporotrichosis is a disease distributed worldwide, there are only a few studies regarding the physiological characteristics of its agent. In general, the optimal temperature for S. schenckii growth is around 30 to 37°C, with growth of all strains being impeded at 40°C (79, 150). Although S. schenckii is able to grow at 35 to 37°C, some growth inhibition is observed compared to that at 28°C. Moreover, this inhibition appears to be geographically related (163).
Several carbohydrates can be assimilated by S. schenckii, such as glucose, fructose, mannose, and cellobiose (79, 200). However, there is some variability in assimilation of sucrose, arabinose, starch, raffinose, and ribitol (79, 150). Starch assimilation is also affected by fungal preservation under some storage methodologies, such as the Castellani method (160). The carbohydrate concentration available during S. schenckii growth modulates melanin synthesis by this fungus (Fig. 3), enhancing pigment formation in a glucose concentration-dependent manner (5). This fungus is not able to ferment any carbohydrate (53).
Glucose concentration-dependent increase of melanin synthesis. The concentration of glucose (%, wt/vol) in each culture is indicated by the numbers on the agar plates. S. brasiliensis strain 17307, grown at 22°C (A), and S. schenckii strain 23250,...
Some physiological differences between the two different S. schenckii morphologies may be observed. Mycelial-phase S. schenckii can grow well at pHs of around 3.0 to 11.5, but yeast cells can grow only within the pH range of 3.0 to 8.5. The yeast phase is also more osmotolerant (30%) than the mycelial phase (20%), as is true for halophila. The mycelial phase withstands growth in 7% NaCl, but the yeast-phase S. schenckii can grow well in 11% NaCl (79).
S. schenckii is able to split urea (79, 150, 233), perform reductive iron acquisition with secreted extracellular enzymes (270), and tolerate cycloheximide at 0.25% (150). Thiamine is required for fungal growth (60, 101).
S. SCHENCKII ECOLOGY
S. schenckii often enters the host through traumatic implantation (126, 129, 170, 196). In nature, the fungus has been found to live as a saprophyte on living and decaying vegetation, animal excreta, and soil (118, 126, 156, 162). Organic material in soil is fundamental for mycelium development. The fungus thrives in soil plentiful in cellulose, with a pH range from 3.5 to 9.4 and a temperature of 31°C (177). The relative humidity cannot be below 92% (126).
It has been proposed that the armadillo Dasypus septemcinctus may be a reservoir of S. schenckii, since armadillo hunting was reported by several patients with sporotrichosis in Uruguay (143). In fact, this armadillo harbors S. schenckii in neither its intestine nor its epidermis, but the fungus can be found on the dry grass used by these animals for nesting (126). Here we find an apparent incongruity between the organisms needing high humidity for growth (126) yet being found on dry grass of armadillo nests. Probably these differences are geographically related, like growth inhibition at high temperatures (163). Moreover, we cannot discard the hypothesis that different species within the S. schenckii complex (150, 151) have different humidity requirements for growth.
There is another armadillo species, Dasypus novemcinctus, which is susceptible to systemic, fatal sporotrichosis (111, 264). Other animals related to S. schenckii transmission are parrots, rodents, cats, dogs, squirrels, horses, and birds (126, 212). Moreover, S. schenckii was also isolated from aquatic animals, primarily fish and dolphins (91, 164), as well as from insects that had been in direct contact with the fungus (126). Some authors have reported sporotrichosis cases due to mosquito bites (129).
It has been confirmed that S. schenckii is associated with plants. Sphagnum moss, rose thorns, and hay are especially recognized to harbor this pathogenic fungus (64, 71, 156). However, S. schenckii does not have the potential to be a plant pathogen, probably because extracts from several plants have antifungal activity against S. schenckii (81, 153, 154, 213). In fact, it has been described that when S. schenckii is inoculated in living or dead sphagnum moss, the fungal cell population proliferates in the moist dead plants but not in live moss (271), suggesting that plants have some mechanism to control S. schenckii overgrowth.
There has been some reports about S. schenckii isolation from food (1, 117). Nevertheless, this fungus does not appear to have the potential to cause food-borne infection (116).
There are some reports on the isolation of S. schenckii from environmental sources. Methods include direct isolation of the fungus by plating the supernatants of samples suspended in water or physiological saline solution with antibiotics in Mycosel agar medium or by inoculation of this suspension in susceptible mice, such as BALB/c, with further culture of spleens, livers, and lungs of the infected animals (60, 162). Direct isolation appears to be more effective to obtain S. schenckii from environmental samples. However, colonies obtained after mouse inoculation have been free of microbial contamination (162).
PUTATIVE S. SCHENCKII VIRULENCE FACTORS
We can define a virulence factor as a feature of a microorganism that allows or enhances microbial growth in the host. To study and characterize these factors, is necessary compare the microbe-host interactions of an isolate that expresses the suspected factor and a mutant isolate that has lost the ability to express it, which can be attained by induced mutagenesis through molecular strategies. If differences in the infections caused by these different isolates are noted, it is imperative to make the mutant isolate recover the ability to express the studied factor and check whether or not the organism then regains the capacity to cause infection similar to that of the parental wild-type strain (100).
Discovery of the origin of microbial virulence has been the main goal of several studies. In general, the most accepted theory is that with microbial interactions with other organisms present in the natural habitat of the pathogen, the microorganisms acquire survival strategies tending to a higher virulence when they accidentally find an animal host. For instance, these microorganisms, in the mammalian host, usually have the ability to form biofilms and mechanisms to acquire iron and produce proteolytic enzymes that will lead to higher virulence (40).
Regarding this theory, Steenbergen and coworkers (240) suggested that the origin of virulence in S. schenckii should be related to the intermicrobial interactions in its environment. The authors demonstrated that when ingested by Acanthamoeba castellanii, a soil amoeba, S. schenckii yeast cells are able to survive within the protozoan, kill it, and use it as nutrient. This behavior is not shared by pathogenic fungi that do not have the soil as habitat, such as Candida albicans, or by fungi that are not primarily pathogenic, such as Saccharomyces cerevisiae. On the other hand, other dimorphic fungal pathogens, such as Histoplasma capsulatum and Blastomyces dermatitidis (240) as well as Cryptococcus gattii, a highly virulent yeast pathogen (148), have the same behavior as S. schenckii when in contact with A. castellanii.
Little is known about S. schenckii virulence factors due to the lack of studies in this field, in part because S. schenckii is not responsive to genetic analysis. However, some putative virulence factors have appeared from some investigations.
One of the putative S. schenckii virulence factors, which is also a virulence factor of other pathogenic fungi, is thermotolerance (40). In fact, isolates able to grow at 35°C but not at 37°C are incapable of causing lymphatic sporotrichosis and produce fixed cutaneous lesions instead. The fungi isolated from lymphatic, disseminated and extracutaneous lesions show tolerance and growth at 37°C (126). Results from a more recent study demonstrate that S. schenckii isolates from Colombia, where most patients are affected with fixed cutaneous sporotrichosis, exhibit high growth inhibition at 35 and 37°C, in contrast to isolates from Mexico and Guatemala, where lymphatic sporotrichosis prevails (163). In an in vivo mouse assay, it has been shown that if the mice have their feet warmed in cages with heat on the floor, progression of sporotrichosis is reduced compared to that in control infected mice maintained in regular cages. Isolates from pulmonary lesions, however, replicate regularly in both groups of mice (246).
Both morphological stages of S. schenckii have the ability to synthesize melanin. This is an insoluble compound highly related to virulence in several fungi (106). Melanin production in S. schenckii dematiaceous conidia occurs through the 1,8-dihydroxynaphthalene (DHN) pentaketide pathway (202). Macroscopically, only the mycelial phase of the fungus is melanized. However, melanin production in yeast cells was demonstrated in vitro during infection (171). Recently, it has been demonstrated that S. schenckii can also produce melanin using phenolic compounds such as 3,4-dihydroxy-l-phenylalanine (l-DOPA) as a substrate both in filamentous and yeast forms (5). It is interesting to note that only conidia can be melanized by the DHN pathway, but if l-DOPA is present, hyphae can be melanized as well (Fig. 4).
Melanin ghosts of S. schenckii 18782 strain under several culture conditions. (A) Cultures on minimal medium at 25°C yield melanin ghosts only from dematiaceous conidia. (B) When l-DOPA is added to minimal medium, both hyphae and conidia are melanized....
Since S. schenckii is a soil-accommodated fungus that does not require host parasitism to complete its life cycle, fungal melanization must be also important against unfavorable environmental conditions, since mycelium is the fungal form encountered in nature (171).
In vitro studies indicate that melanization in S. schenckii is controlled by several factors, such as temperature, pH, and nutrient conditions (5). Moreover, similar culture media from different suppliers can yield differences in melanization within a single S. schenckii strain (250). It has been shown that conidial melanization enhances S. schenckii resistance to macrophage phagocytosis, allowing the first steps of infection, since these structures usually are the fungal infective particles (202). Corroborating this hypothesis, it has been demonstrated by molecular typing of an S. schenckii strain isolated from a laboratory worker who had handled a pigmented strain and an albino strain of S. schenckii that the isolate from the patient had the same genotypic profile as the dematiaceous strain (48).
Melanization also has a role in the pathogenesis of cutaneous sporotrichosis, since pigmented isolates had a greater invasive ability than the albino mutant strain in an experimental rat model of sporotrichosis. The albino strain also was restricted to the core of the granuloma. In addition, the melanized strain promoted the formation of multifocal granulomas (145).
Some S. schenckii melanization in vivo has been previously described, such as a weak brown halo on the yeast S. schenckii cell wall when infected sporotrichosis tissues are stained with Fontana-Masson stain, a technique initially developed to demonstrate melanin in C. neoformans (127). This hypothesis has now been confirmed by detection of Sporothrix melanin ghosts in tissues from infected animals as well as by detection of antimelanin antibody in sera from patients with sporotrichosis (5, 171). Since melanization decreases the susceptibilities of H. capsulatum and C. neoformans to amphotericin B and caspofungin (261), melanin pigment in S. schenckii may hamper treatment in some sporotrichosis cases, especially in cases of extracutaneous disease or in patients infected with human immunodeficiency virus (HIV) (171). However, there have been no studies confirming this hypothesis.
Primary adhesion to endothelial and epithelial cells as well as on extracellular matrix components is essential to an effective invasion of host tissues by pathogens. Both conidia and yeast cells from S. schenckii are able to recognize three important glycoproteins from the extracellular matrix: fibronectin, laminin, and type II collagen (134, 135). Some studies have demonstrated that the fungus has integrins or adhesin lectin-like molecules that recognize human fibronectin at several points on the molecule (135). The fibronectin adhesins are located on the surface of yeast cells, and their expression is related to fungal virulence (251). It is also known that these fibronectin receptors are different from laminin receptors (133). These receptors are present on both hyphae and fungal yeasts, although yeasts have a greater ability to bind to the extracellular matrix. The existence of these adhesins would favor adherence to host tissues and fungal dissemination throughout the body (133). Expression of these molecules in S. schenckii is probably related to virulence, since their preferential expression is in the parasitic rather than the saprophytic form of the fungus. Recently a 70-kDa glycoprotein from an S. schenckii isolate was described, and its participation in adhesion to the dermal extracellular matrix was demonstrated (206).
This fungal pathogen is also able to interact in vitro with human endothelial cells, which can internalize fungal yeast cells without injury or decreased viability. Moreover, the fungus can also cross the intercellular space. Both processes facilitate fungus bloodstream penetration and consequent hematogenous dissemination (69). Transendothelium migration occurs through a paracellular route involving extracellular matrix proteins, in a process mediated by transforming growth factor β1 (TGF-β1) (70). Although the endothelial proteins responsible for this interaction have been characterized, fungal proteins needed for recognition of and adhesion to these cells are unknown, and their part in fungal virulence requires clarification.
Sgarbi and coworkers, analyzing lipids from S. schenckii through spectroscopic methods, have identified ergosterol peroxide from S. schenckii yeast cells. This compound can be converted to ergosterol when in contact with an enzyme extract from the fungus. The ergosterol peroxide, found in a pathogenic fungus for the first time in S. schenckii, is formed as a protective mechanism to evade reactive oxygen species during phagocytosis and may also represent a virulence factor. Apparently, however, survival of virulent S. schenckii yeast cells after phagocytosis of polymorphonuclear host cells relies on other detoxification strategies besides the one leading to ergosterol peroxide synthesis (32, 231).
Proteins Related to Virulence
Roles of diverse proteins in the virulence of different fungal pathogens have been described. For instance, the Paracoccidioides brasiliensis immunodominant antigen, a glycoprotein of 43 kDa, is the molecule responsible for laminin and fibronectin recognition and binding, which increase fungal virulence (158, 262). Calcium binding proteins are important in H. capsulatum virulence, enabling acquisition of this ion in environments with calcium limitations (265). A series of virulence-related proteins, such as different adhesins, have been described for Aspergillus fumigatus, including a 30-kDa hemolysin containing several proteases that favor pulmonary colonization and destruction of effective humoral molecules and a 350-kDa catalase needed for phagocytosis survival (131). However, the function of different S. schenckii proteins in virulence is still unclear. It is believed that acid phosphatases act on fungus-macrophage interactions, although no definite evidence to support this theory exists (100). Peptido-rhamnomannans of the fungal cell wall cause depression of immune response until the sixth week of infection and may act as a virulence factor (34). An antigenic preparation from the S. schenckii yeast phase shows proteolytic activity against different human IgG subclasses, suggesting that some secreted proteins may interfere with the immune response of the host (204). Due to the lack of information, characterization of S. schenckii proteins and determination of new virulence factors are imperative for a better understanding of sporotrichosis pathogenesis.
IMMUNE RESPONSE IN SPOROTRICHOSIS
The virulence of S. schenckii is one of the factors thought to play a role in the development of sporotrichosis (32), but there are discordant results concerning disease evolution in experimental sporotrichosis with S. schenckii clinical isolates from cutaneous and disseminated infection (29, 176), indicating that host immune responses also substantially participate in the progress of sporotrichosis (32).
The immunological mechanisms involved in prevention and control of S. schenckii infections are still not very well understood. However, they probably include both humoral and cellular responses (32, 33, 147), which appear to be triggered by distinct antigens. Surface cell antigens, especially some lipids, inhibit the phagocytosis process, while the humoral response is induced by secreted fungal proteins, the exoantigens, which are not involved in the cellular response (35). The innate immune response also plays a role in the pathogenesis of sporotrichosis (32).
A complement system can be activated by S. schenckii, especially the alternate pathway, although classic complement activation cannot be excluded. Complement activation may support fungal yeast cell phagocytosis by C3b component deposition on the fungal cell wall. The membrane attack complex also contribute to fungal cell lysis (230, 255).
Recent studies have emphasized the importance of Toll-like receptor 4 (TLR4) in sporotrichosis. TLR4, also designated CD284, is an important molecule involved in the activation of the innate immune system that, in sporotrichosis, is able to recognize molecules within a lipid extract from the yeast form of the fungus. This interaction leads to the induction of an oxidative burst against the fungus (32).
Acquired immunity against the fungus requires the action of activated macrophages. They can be activated during sporotrichosis by CD4 T lymphocytes, which release gamma interferon (IFN-γ), a strong macrophage activator (247), and by other antigen-presenting cells, establishing a link between innate and adaptive immune responses (32). Tumor necrosis factor alpha (TNF-α), a cytokine that acts on activated macrophages to produce nitric oxide (35), an antioxidant product presenting a high cytotoxic effect against S. schenckii (66), is produced upon incipient and terminal infection, hopefully providing total resolution (147). Although nitric oxide is a fungicidal molecule, this compound may be implicated in immunosuppression in vivo, because high levels of TNF-α and NO released after yeast dissemination into the tissues lead to the induction of molecules suppressing T cell responses, such as interleukin10 (IL-10), FasL, and CTLA-4. This deleterious effect of NO occurs just upon initial infection, becoming crucial some time after fungal inoculation (68). In fact, TNF-α production drastically subsides at 4 to 6 weeks after experimental infection, inducing the fungus to reproduce and infect host tissues. The opposite situation occurs 2 months after infection, when the levels of IL-1 and TNF-α increase, favoring fungal elimination (36).
After the phagocytosis of S. schenckii conidia and yeast cells, monocytes and macrophages are also strongly induced to produce reactive oxygen species (202). These reactive species, especially superoxide anion and its oxidative reactive metabolites, which are also produced by neutrophils, are involved in fungistatic and fungicidal responses, and their absence is related to a higher lethality in mouse experimental infections (108). Therefore, the Th1 response is of great importance in sporotrichosis pathogenesis, acting as the key factor in controlling fungal infection and with its differential activation leading to varied clinical manifestations of the disease (258). Similar observations on the activation of Th1 cells have led to them being seen as being responsible for different clinical manifestations in other cutaneous infectious diseases, such as leishmaniasis (38).
The humoral immune response is driven by IL-4 produced by Th2 cells. In experimental sporotrichosis, IL-4 release is enhanced at 5 to 6 weeks after infection (147), suggesting the participation of the humoral immune response only in the advanced stages of sporotrichosis (32). Antibodies may have some effect on S. schenckii development, since a monoclonal antibody against a glycolipid antigen is able to hinder S. schenckii growth and differentiation in vitro (254). A monoclonal antibody against the 70-kDa adhesin is also protective in murine model of sporotrichosis (174). Nonetheless, little is known about antibodies elicited during the course of sporotrichosis. It has been described that mice infected with S. schenckii are able to produce specific IgG1 and IgG3 antibodies against a 70-kDa fungal protein during experimental infection, with these antibodies perhaps being related to fungal elimination in these organisms (173). During human sporotrichosis, our group has demonstrated the production of IgG, IgM, and IgA antibodies against mycelial-phase S. schenckii exoantigens. Nevertheless, since patients with different clinical forms of sporotrichosis produce similar amounts of these antibodies, we believe that the humoral immune response against proteins secreted by S. schenckii does not play a role in sporotrichosis pathogenesis (6).
Sporotrichosis can be diagnosed through a correlation of clinical, epidemiological, and laboratory data. Laboratory analysis for the determination of sporotrichosis includes direct examination of specimens such as tissue biopsy specimens or pus from lesions. In case of disseminated infections, other specimens, such as sputum, urine, blood, and cerebrospinal and synovial fluids, can be analyzed, depending on the affected organs.
Direct examination of specimens is usually conducted with 10% potassium hydroxide in order to observe parasitic budding yeast cells. These yeasts are small (2 to 6 μm in diameter) and scarce and consequently are difficult to detect upon direct examination of specimens collected from humans. However, when the same test is performed with samples collected from infected cats, due to the high fungal burden in these animals, yeast cells can be easily found, even at a magnification of ×400 (Fig. 5). Fluorescent-antibody staining can help in the observation of yeast forms of S. schenckii; however, this is not a technique that is readily available in most laboratories (130), especially in underdeveloped countries. When the Gram stain is used on the clinical material, yeast cells appear positively stained, sometimes within giant cells or polymorphonuclear lymphocytes (129). For the detection of S. schenckii, some authors recommend Giemsa stain after 10 to 15 dilutions of pus in physiological solution (12, 129). These staining procedures also lack sensitivity. Observation of yeast cells through direct examination, however, is not conclusive for sporotrichosis diagnosis. The characteristic “cigar-shaped” buds (2 by 3 to 3 by 10 μm) are not always witnessed. Moreover, yeast cells of H. capsulatum and Candida glabrata may be misidentified as S. schenckii (130).
Direct examination of clinical specimens for diagnosis of sporotrichosis. (A) KOH mount of a tissue fragment from a cat with sporotrichosis, showing cigar-shaped (arrow) and budding (dashed arrow) S. schenckii yeast cells. Note the high fungal burden...
Direct examination of pus obtained from lesions of patients with sporotrichosis, without potassium hydroxide, also permits the detection of asteroid bodies. These structures were confirmed in 43.75% of patients, and the sensitivity of the examination can be enhanced (up to 93.75%) if the initial pus is discarded and new samples are collected more deeply. This can help to initiate specific treatment before the results of the culture examinations are available (78).
Although S. schenckii may be seen in tissue with the routinely used hematoxylin and eosin (H&E) stain, other special stains such as Gomori methenamine silver (GMS) or periodic acid-Schiff (PAS) stain can be employed to enhance fungal detection (130, 170). Fontana-Masson staining is negative (54). Atypical S. schenckii cells can appear spherical and surrounded by a PAS-positive capsule, resembling Cryptococcus cells (126). Once again, parasitic cells of S. schenckii are difficult to visualize due to the paucity of yeasts in lesions from humans (170) or other animals such as dogs (57).
Tissue reaction must be also evaluated in histopathological examinations from patients with sporotrichosis. S. schenckii usually causes a mixed suppurative and granulomatous inflammatory reaction in the dermis and subcutaneous tissue, frequently accompanied by microabscess and fibrosis. Cutaneous infections may also exhibit hyperkeratosis, parakeratosis, and pseudoepitheliomatous hyperplasia (130). Foreign bodies of vegetal origin related to the traumatic inoculation of the agent may also be encountered (182).
Besides intact polymorphonuclear cells, granulomas in sporotrichosis usually contain cellular debris, caseous material, giant and epithelioid cell lymphocytes, plasmocytes, and fibroblasts as well as S. schenckii yeast cells within phagocytic cells or in the extracellular medium (129). Miranda and collaborators (167) reported that in dogs with sporotrichosis, lesions present well-formed granulomata, with marked neutrophil infiltration. The peripheral infiltrate often is devoid of lymphocytes and macrophages. Taken together, this information enables the differentiation of sporotrichosis and leishmaniasis in these animals.
Some histopathological alterations, such as presence or predominance of epithelioid granulomas, presence of foreign body granulomas, predominance of lymphocytes, presence or predominance of caseous necrosis, and predominance of fibrinoid necrosis and fibrosis, are related to the lack of observation of the fungus in tissue sections from human patients. When the fungus is not in evidence, suppurative granulomas, neutrophils, and liquefaction necrosis are uncommon (187).
Splendore in 1908 described a radiate eosinophilic substance in human tissues from patients with sporotrichosis, and Hoeppli in 1932 reported an eosinophilic material around schistosome larvae (126). The Splendore-Hoeppli reaction is indicative of a localized immunological host response to antigens of diverse infectious organisms, including fungi, bacteria, and other parasites. It appears as radiating homogenous, refractile, eosinophilic clublike material surrounding a central eosinophilic focus (130). There are several reports concerning asteroid bodies, the Splendore-Hoeppli reaction in sporotrichosis, in histopathological tissue sections from sporotrichosis patients, ranging in positivity from 20 to 66% (78). Other authors, however, report an absence of this structure in analyzed samples (187). Yeast cells remain viable inside the asteroid bodies, which present IgG and IgM from the host on the spikes of the radiated crowns, suggesting that asteroid bodies are resistance structures which use immune molecules of the host to advantage the yeasts (203).
Definitive sporotrichosis diagnosis is based on the isolation and identification of the etiological agent in culture (126). Isolation of S. schenckii is easily obtained after spreading of the clinical specimens on Sabouraud agar with chloramphenicol and on media with cycloheximide, such as mycobiotic agar. After 5 to 7 days of incubation at 25°C, filamentous hyaline colonies start to grow, and after some time, they may develop a dark color, usually in the centers of the colonies (170). To identify an isolate as S. schenckii, one must demonstrate that it undergoes dimorphism by subculturing the fungus on enriched media such as brain heart infusion agar, chocolate agar, and blood agar at 35 to 37°C for 5 to 7 days. Occasional isolates can be difficult to convert and may require multiple subcultures and extended incubation (216). After S. schenckii conversion to the yeast phase, colonies acquire a creamy aspect and a yellow to tan color (170). Environmental Sporothrix strains may also form the yeast phase when grown on appropriate media at 37°C. For this reason, observation of dematiaceous conidia in colonies maintained at 25°C is mandatory (54, 60, 216). For this purpose, slide culture preparations with potato dextrose agar or cornmeal agar are ideal to study S. schenckii conidiogenesis (54).
Positive cultures provide the strongest evidence for sporotrichosis, allowing diagnosis of almost all cases of cutaneous disease. Nevertheless, culture diagnosis has significant limitations, mainly in some manifestations of the disease such as S. schenckii induced arthritis, where the collection of material for culture is difficult.
Nonculture methods have been developed to improve the rate and speed of mycological diagnosis (194, 241). Molecular detection of S. schenckii is useful for a rapid diagnosis of sporotrichosis and also valuable in cases of negative cultures due to low fungal burden or secondary infections.
Up to now, there has been a scarcity of molecular methods for the detection of S. schenckii DNA from clinical specimens. Sandhu and collaborators (211) reported the development of 21 specific nucleotide probes targeting the large-subunit rRNA genes from several fungi, including S. schenckii. The authors adopted a protocol for DNA extraction from clinical specimens that consists of boiling the specimens in an alkaline guanidine-phenol-Tris reagent, followed by amplification of a variable region of the 28S rRNA gene with universal primers and amplicon identification using the specific probes. The results displayed a high level of specificity for this test.
Some methodologies to identify S. schenckii colonies from pure cultures have been described. Specific probes for fungi with yeast-like morphology in vivo, including all dimorphic fungal pathogens, were developed for the detection of PCR amplicons in an enzyme immunoassay format. S. schenckii DNA was able to hybridize to the probe to detect all dimorphic fungi as well as to its specific probe (137). Specific oligonucleotide primers based on the chitin synthase gene were also developed. This primer was able to detect 10 pg of genomic S. schenckii DNA (110). Primers to distinguish S. schenckii from related species such as Ceratocystis stenoceras, based on the DNA topoisomerase II genes, permitted the amplification of fragments of 663 to 817 bp from S. schenckii and a 660-bp fragment from S. schenckii var. lurei. Another set of primers allowed the amplification of a specific 305-bp fragment from S. schenckii var. lurei (109). These detection systems may be useful as diagnostic tools for the detection of human and animal sporotrichosis. In fact, a PCR assay based on the internal transcriber space in the rRNA gene has been used for the identification of an S. schenckii strain from an atypical case of sporotrichosis (75).
Sporotrichin Skin Test
The cutaneous sporotrichin skin test detects delayed hypersensitivity, i.e., the cellular immune response, and can be a useful diagnostic tool, but its major usefulness is in epidemiological investigations. This reaction is usually positive in about 90% of confirmed sporotrichosis cases but can also indicate previous infection with the fungus (105). The sporotrichin skin test has been successfully applied to confirm the diagnosis of bulbar conjunctival sporotrichosis after the pathological examination revealed yeast-like cells (113).
Epidemiological studies usually involve the sporotrichin skin testing of individuals living or working in a determinate area together with attempts to isolate the fungus from the soil in that area. For instance, this test gave 6.25% positivity in a Mexican state where virulent strains of S. schenckii were isolated from soil (210). On the other hand, 13.67% positivity was found among healthy mine workers from Brazil, although the fungus was not isolated from soil samples from the mines investigated (198).
Despite the current use of the sporotrichin skin test in several studies throughout the world, the antigen adopted in these tests lacks standardization. Several studies on sporotrichin in Brazil were performed with a 5 McFarland standard suspension of heat-killed yeast cells (129). A retrospective 10-year study in Mexico used extracted mycelial antigens at a 1:2,000 dilution (28), while another Mexican study diluted yeast-phase antigens at 1:4,000 (210). These variations in antigen production may lead to differences in results.
Several methodologies have been described for the immunological diagnosis of sporotrichosis based on antibody detection in sera from infected patients. Precipitation and agglutination techniques were first adopted. Double immunodiffusion for sporotrichosis does not usually show cross-reactions with sera from patients with chromoblastomycosis or leishmaniasis, infectious diseases with similar clinical manifestations. Immunoelectrophoresis has also been employed, with an anodic arc, called S arc, being observed in all positive cases (2). Tube agglutination and latex agglutination have been utilized for sporotrichosis serodiagnosis since the 1970s, and very good sensitivity (96% and 94%, respectively) and specificity (98 and 100%, respectively) have been observed (26, 41, 112). These tests, however, lack sensitivity in cases of cutaneous sporotrichosis (2, 196) and do not permit the determination of the immunoglobulin isotype involved in the response.
Immunoenzymatic assays are currently being used more frequently for serodiagnosis purposes. The publication of an immunoblot assay for serodiagnosis of sporotrichosis dates back to 1989, when molecules of 40 and 70 kDa from exoantigen preparations from the S. schenckii yeast form showed 100% sensitivity and 95% specificity (229
Sporotrichosis associated with zoonotic transmission remains a relevant public health problem in Rio de Janeiro, Brazil, affecting a large at-risk population, which includes HIV-infected individuals. We assessed patients co-infected by Sporothrix spp. and HIV over time in the context of an unabated sporotrichosis epidemic.
A retrospective cohort retrieved information from a National reference institute for infectious diseases regarding 48 patients with sporotrichosis-HIV co-infection (group 1) as well as 3,570 patients with sporotrichosis (group 2), from 1987 through March 2013. Most patients from group 1 were male (68.8%), whereas women were predominant in group 2 (69.1%; p<0.0001). Patients from group 1 were younger than those from group 2 (μ = 38.38±10.17 vs. 46.34±15.85; p<0.001) and differed from group 2 in terms of their race/ethnic background, with 70.8% non-white patients in group 1 vs. 38.6% from group 2 (p<0.0001). Close to half (∼44%) of the patients from group 1 were hospitalized due to sporotrichosis over time, whereas hospitalization was very unlikely in group 2, among whom approximately 1% were hospitalized over time. Dissemination of sporotrichosis was the main cause of hospitalization in both groups, although it was more common among hospitalized patients from group 1 (19/21 [90.5%] vs. 16/37 [43.2%]; p<0.001). Over the period under analysis, eight patients died due to sporotrichosis (3/48 vs. 5/3,570). The diagnosis of sporotrichosis elicited HIV testing and subsequent diagnosis in 19/48 patients, whereas 23/48 patients were simultaneously diagnosed with the two infections.
HIV infection aggravates sporotrichosis, with a higher incidence of severe disseminated cases and a higher number of hospitalizations and deaths. Underserved populations, among whom sporotrichosis has been propagated, have been affected by different transmissible (e.g., HIV) and non-transmissible diseases. These populations should be targeted by community development programs and entitled to integrated management and care of their superimposed burdens.
Sporotrichosis is a subcutaneous, worldwide-distributed mycosis, endemic in some areas and is caused by dimorphic fungi from the complex Sporothrix schenckii. Its association with zoonotic transmission remains a relevant public health problem in Rio de Janeiro, Brazil, affecting a large at-risk population, which includes HIV-infected individuals. A comprehensive search of a National reference institute for infectious diseases' database retrieved information regarding 48 patients with sporotrichosis-HIV co-infection (group 1), as well as 3,570 patients with sporotrichosis (group 2), registered from 1987 through March 2013. Group 1 mainly comprised young, non-white men, while group 2 was predominantly comprised of white middle-aged women. HIV infection aggravates sporotrichosis, as seen with patients from group 1, who presented more severe disseminated sporotrichosis, a higher need for hospitalization and risk of death due to this mycosis. Due to its aggressive presentation, sporotrichosis elicited HIV testing and subsequent diagnosis in 19/48 patients. Underserved populations, among whom sporotrichosis has propagated, have been affected by different transmissible (e.g., HIV) and non-transmissible diseases. These populations should be targeted by community development programs and entitled to integrated management and care of their superimposed burdens.
Citation: Freitas DFS, Valle ACFd, da Silva MBT, Campos DP, Lyra MR, de Souza RV, et al. (2014) Sporotrichosis: An Emerging Neglected Opportunistic Infection in HIV-Infected Patients in Rio de Janeiro, Brazil. PLoS Negl Trop Dis 8(8): e3110. https://doi.org/10.1371/journal.pntd.0003110
Editor: Joseph M. Vinetz, University of California San Diego School of Medicine, United States of America
Received: February 15, 2014; Accepted: July 12, 2014; Published: August 28, 2014
Copyright: © 2014 Freitas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Financial support was received from FAPERJ/Rio de Janeiro, Brazil (grant proc. E-26/110.619/2012) and PAPES VI – CNPq/Fiocruz (grant proc. 407693/2012-2). DFSF received financial support from CNPq and CAPES. RMZO was supported in part by CNPq (350338/2000-0) and FAPERJ (E-26/103.157/2011). MCGG received partial funding from the Brazilian National STD/AIDS Program, Ministry of Health (46/CV079/2006). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Sporotrichosis is a subcutaneous mycosis with a worldwide distribution that is endemic in some areas of Latin America. The infection is caused by a dimorphic fungus previously described as a single species, Sporothrix schenckii, that is now understood as a complex of different species of clinical interest . Molecular studies have identified Sporothrix globosa, Sporothrix mexicana, Sporothrix brasiliensis and S. schenckii as responsible for sporotrichosis in different regions –. The classical infection is associated with traumatic subcutaneous inoculation of soil, plants, and organic matter contaminated with fungus, with rare cases of transmission from infected animals . Most patients with sporotrichosis have a localized disease limited to the skin and subcutaneous tissue (lymphocutaneous and cutaneous fixed forms), comprising up to 95% of cases. Dissemination to various organs and systems occurs in rare cases, mainly in immunosuppressed individuals .
In Rio de Janeiro state, Brazil, sporotrichosis has become an urban endemic/epidemic phenomenon, with transmission from infected cats to humans in ∼91% of human cases . These cases came from the greater metropolitan area of Rio de Janeiro (the capital city of Rio de Janeiro state), forming a sporotrichosis belt. These areas are low-income, underserved areas, with scarce and inadequate health services –.
The increase in the number of cases of the disease has been continuous for more than 15 years and remains on the rise, affecting vulnerable groups of humans, as well as domestic and stray cats . In 2012, Freitas et al.  described the clinical manifestations and evolution of sporotrichosis in human immunodeficiency virus (HIV)-infected patients in the largest case series reported to date worldwide.
In Brazil, the HIV/AIDS epidemic has been stable and concentrated in some urban areas, mostly affecting men who have sex with men and female sex workers. The overall epidemic dynamics have switched from a population of higher socioeconomic status to individuals from low-middle and lower socioeconomic strata . These dynamics favor a superposition of HIV spread with other infections such as tuberculosis and leprosy, which have been uniquely prevalent in contexts of poverty and pronounced socioeconomic and social geographic inequality , .
The present study summarizes data from a large dataset of sporotrichosis cases, consisting of 3,570 patients registered from 1987 up to March 2013, as well as 48 patients co-infected by HIV and sporotrichosis, who sought care at a reference infectious disease unit located in Rio de Janeiro, Brazil. Based on our previous clinical data  our hypothesis is that patients co-infected by HIV with low CD4+ cell count are prone to worse outcomes as sporotrichosis evolves as an opportunistic condition.
The Research Ethics Committee of the Instituto de Pesquisa Clínica Evandro Chagas (IPEC)/Fundação Oswaldo Cruz (Fiocruz), RJ, Brazil, approved this study under the protocol number 0001.0.009.000-06. All patients involved were anonymized for privacy and ethics purposes.
IPEC is a National reference center for infectious diseases. Since the beginning of the sporotrichosis epidemic in Rio de Janeiro in 1998, this center has been the main referral center for the treatment of this mycosis in the state due to its certified laboratory and the optimal infrastructure of its clinical and ancillary services. All services are delivered free of charge, and referral is agile. Patients may be referred to IPEC from any health service (public or private) or may spontaneously seek care. In addition, the AIDS program at IPEC began in 1986 and is currently one of the largest providers of primary, specialty, and tertiary care for HIV-infected individuals and AIDS patients in Rio de Janeiro State.
For this retrospective cohort study, a systematic search of IPEC's clinical database was conducted to identify cases of sporotrichosis-HIV co-infection that were registered from 1987 through March 2013, as well as sporotrichosis cases, overall. All patients diagnosed with sporotrichosis confirmed by laboratory tests were included, as well as patients living with HIV under follow-up in the institute's cohort. Patients with sporotrichosis co-infected with HIV (hereafter denominated “group 1”) and patients with sporotrichosis (“group 2”) constituted the groups under analysis. Additional analyses were conducted on patients from the IPEC HIV/AIDS cohort who were diagnosed with the following opportunistic mycoses: histoplasmosis, cryptococcosis and paracoccidioidomycosis. Only patients aged 18 years old or more at the time of registration were included in the study.
The diagnosis of HIV infection followed Brazilian Ministry of Health regulations, which are summarized as follows: an immune-enzymatic method (ELISA) test plus immune-fluorescence or western blot. HIV serology was conducted in all cases of disseminated cutaneous and disseminated cases of sporotrichosis or evidence of HIV signs or symptoms. A non-paired random HIV test was performed with the stored blood samples of 850 patients from group 2 who were registered from 2000 through 2008.
Isolation of Sporothrix spp. from clinical specimens was used as the study's key inclusion criterion, as previously described by Barros et al. . When the patient had clinical or laboratory signs of HIV-related immunodeficiency (CD4+ count <200 cells/µL), fungal dissemination was investigated by culturing his/her sputum, blood, urine, and cerebrospinal fluid (CSF) samples, as well as by endoscopic and imaging studies, as previously described by Freitas et al. . Clinical cases of sporotrichosis were classified as localized (lymphocutaneous and fixed forms), cutaneous disseminated and disseminated forms. This last form may involve extracutaneous tissues such as the skeletal system, lungs, testis, nervous system, and mucous membranes .
Demographic data included the following categories: gender, age, ethnicity/color, city of residence and education. Ethnicity/color was established as white or non-white (brown [mulatto] and black were grouped together here) by the administrative staff at the time of registration in the institute until the year 2005, after which this information was then self-reported by the patients. The clinical characteristics of sporotrichosis, as well as the associated morbidity and mortality, were summarized by the variables as follows: “date of diagnosis of HIV infection”; “HIV plasma viral load”; “CD4+ cell count” and “use of highly active antiretroviral therapy (HAART) for patients from group 1; “date of diagnosis of sporotrichosis”; “hospitalization due to sporotrichosis” (yes/no); main cause for hospitalization among those hospitalized as a consequence of sporotrichosis (dissemination, secondary bacterial infection, hypersensitivity reaction [erythema multiforme or erythema nodosum], and local worsening); comorbidities (cardiovascular disease [ICD-10:I51.6], diabetes [ICD-10:E10–E14], chronic obstructive pulmonary disease (COPD) [ICD-10:J44], and alcoholism [ICD-10:F10]); number and length of hospitalizations due to sporotrichosis; as well as deaths secondary to sporotrichosis.
Additional analyses took into consideration the year of occurrence of opportunistic mycoses (histoplasmosis, cryptococcosis and paracoccidioidomycosis) in patients from the IPEC HIV/AIDS cohort.
Sociodemographic, clinical and laboratory data were entered into contingency tables and cross-compared using parametric and non-parametric tests (e.g., chi-square or Fisher's exact tests for categorical variables, and t-tests or the Wilcoxon-Mann-Whitney test for means for continuous variables). A p-value lower than 0.05 was defined as statistically significant for the sake of our analyses. Analyses were carried out with the help of SPSS (17.0), R (version 2.15.3) and Microsoft Office Excel 2013.
From 1987 through March 2013, 3,618 patients were diagnosed with sporotrichosis at IPEC and 48 of them were co-infected with HIV (Figure 1). The main sociodemographic aspects of the patients with sporotrichosis are summarized in Table 1.
Figure 1. Annual number of patients with sporotrichosis at IPEC from 1987 through March 2013.
A) Sporotrichosis and HIV (group 1) and B) annual proportion of patients diagnosed with sporotrichosis among all patients diagnosed at IPEC.
When cross-comparing patients from groups 1 and 2, some interesting differences were evident. Individuals from both groups clustered in the same geographic area, i.e., the outskirts and impoverished neighborhoods of the metropolitan region of Rio de Janeiro (95.8% and 97.2%, respectively) and had a similar low educational background. Among the patients from group 1, approximately 40% of patients had more than 8 years of schooling and a slightly higher proportion of patients from group 2 (50.6%) had a similar educational level (this difference was not statistically significant; p>0.2).
Most patients from group 1 were male (68.8%), whereas women predominated in group 2 (69.1%; p<0.0001). Patients from group 1 were younger than those from group 2 (mean age = 38.38±10.17 years vs. 46.34±15.85 years; p<0.001) and differed from those from group 2 in terms of their race/ethnic background, with 70.8% non-whites in group 1 vs. 38.6% from group 2 (p<0.0001).
Sporotrichosis has been associated with some major harms and risks. There were 69 hospitalization events due to sporotrichosis among 58 patients (i.e., some of them were hospitalized more than once; Table 2). However, the proportion of patients who required hospitalizations over time markedly differed between groups. Close to half (∼44%) of the patients from group 1 were hospitalized over time, whereas hospitalization was a very unlikely event among patients from group 2, among whom approximately 1% were hospitalized over time as a consequence of conditions directly or indirectly associated with sporotrichosis. In addition to the fact that they were much more frequent, hospitalizations were longer among patients from group 1 compared to group 2 (37 days vs. 21 days; not statistically significant, as expected for such small figures). Among patients from group 1, the need for hospitalization due to sporotrichosis was 42 times higher than among those from group 2. This difference becomes even more pronounced (greater than 50 times higher) when successive hospitalizations to perform complex or extensive diagnostic and therapeutic procedures, such as parenteral antifungal treatment, supportive therapy and its associated monitoring, are taken into consideration (Table 2).
Dissemination of sporotrichosis was the main cause for hospitalization in both groups, although it was more common among hospitalized patients from group 1 (19/21 [90.5%] vs. 16/37 [43.2%]; p<0.001). These hospitalized patients from group 1 had a mean CD4+ count of 125 cells/µl (range: 7–323 cells/µl), a median viral load of 4,967 copies/ml (range: <50 [detection threshold] - >500,000 copies/ml [values on the right side of the curve were collapsed into this category]), and only 3 were under HAART in a regular basis, while the other 18 patients were not under this specific treatment (not prescribed, not adherent or on HAART for less than 3 months). On the other hand, local and/or hypersensitivity manifestations of sporotrichosis were predominant in hospitalized patients from group 2 (p<0.001). At least one comorbidity was present in 54.1% (20/37) of the hospitalized patients from group 2, but this was a relatively rare event (3/21 [14.3%]) among hospitalized patients from group 1 (p<0.01) (see Table 2). This difference was mainly due to cardiovascular diseases (p<0.05), whereas the prevalence of diabetes was relatively similar in both groups. Three hospitalized patients belonging to group 1 had other opportunistic infections at the time of hospitalization: one patient had pulmonary tuberculosis, another patient had cryptococcal meningitis and another had cytomegalovirus retinitis.
During the period under analysis, eight patients died due to sporotrichosis (3/48 vs. 5/3,570); thus, death attributed to sporotrichosis occurred 45 times more frequently in patients from group 1.
Sporotrichosis elicited HIV testing and subsequent diagnosis, due to its severe clinical presentation, in 19 patients who were unaware of their HIV status. Four other patients were simultaneously diagnosed with the two infections. However, three of them presented localized disease and HIV-related conditions (chronic seborrheic dermatitis, herpes zoster, weight loss and dyspnea) and one had sporotrichosis exclusively, with lymph node involvement a few days after the diagnosis of the HIV infection. In total, 23 patients were simultaneously diagnosed with the two infections. Among the remaining 25 patients, 13 were under follow-up in the HIV/AIDS cohort and 12 were referred by other HIV/AIDS clinical providers.
In the non-paired random HIV testing performed among 850 patients from group 2, 1 sample was positive (0.12%).
In an effort to better understand the role of sporotrichosis vis-à-vis other endemic or classic opportunistic mycoses affecting patients with HIV/AIDS belonging to the IPEC HIV/AIDS cohort, an additional search of the institution's database was performed. It included 5,385 patients living with HIV/AIDS and focused on diagnoses of histoplasmosis, cryptococcosis and paracoccidioidomycosis reported among patients living with HIV/AIDS since 1987 (Table 3, see also Supporting Information S1).
In recent years, cases of sporotrichosis have been on the rise, whereas figures for histoplasmosis, cryptococcosis and paracoccidioidomycosis have been low or declining (Table 3).
The first case of sporotrichosis and HIV co-infection diagnosed at IPEC was reported in 1999, roughly coinciding with the emergence of sporotrichosis as a public health issue in Rio de Janeiro . Since then, the increase in the number of patients with this co-infection was roughly proportional to the overall increase in the number of sporotrichosis cases over time, with the exception of 2005. During this year, the specialized outpatient service's staff was dramatically reduced by a combination of factors. This anomaly certainly biased our time series, the clientele's demands that year could not be properly addressed. As of early 2006, the service regained its full working capacity.
A greater than proportional increase in patients with sporotrichosis co-infected with HIV has been documented in recent years. This may be explained by an actual acceleration of the propagation of sporotrichosis in the last seven years, by a comprehensive HIV screening by clinicians aware of the possibility of co-infection and the seriousness of this type of double health burden, or a combination of both factors. A possible bias to be pointed is the fact that the most severe cases are prone to be referred to IPEC even more frequently than the regular cases of sporotrichosis. In the context of the stability of the HIV/AIDS epidemic, it is unlikely that a local outbreak of HIV has been taking place among people with sporotrichosis. The modest prevalence of other fungal co-infections and their decline in recent years speak in favor of a unique pattern followed by sporotrichosis, which may or may not be associated with HIV/AIDS.
According to the norms issued by the Brazilian Ministry of Health, sporotrichosis does not constitute a condition for which provider-initiated testing and counseling for HIV is mandatory or strongly recommended (such as for patients diagnosed with tuberculosis). As the clinical forms of sporotrichosis in HIV-infected patients varied according to the patients' immune status, we might be missing asymptomatic seropositive patients from group 2 who would present benign forms of sporotrichosis (lymphocutaneous and fixed), which correspond to ∼90% of the clinical presentation of sporotrichosis cases among our patients overall , . Because of this possibility, we performed a non-paired random HIV testing in approximately one-quarter of the blood samples collected from patients form this group. Despite the limitations intrinsic to this type of strategy (besides the fact that this strategy is the only one that could be accomplished retrospectively), the low prevalence (0.12%) speaks in favor of a modest degree of misclassification (i.e., people assigned to group 2 who actually belong to group 1). Obviously, misclassification constitutes a bias that may compromise any cross-comparative analysis. In this specific study, one tends to overestimate harms and risks associated with co-infection because cases who did not present any evident clinical problem tend to be erroneously included in group 2.
The sociodemographic characteristics of group 1, which is composed of a majority of young males, differ from group 2, which is mostly composed of middle-aged women engaged in domestic duties as previously described in this epidemic , . This finding may reflect the dynamics of the HIV/AIDS epidemic in Brazil. Recent studies have shown that the number of affected men is still increasing, especially among young men who have sex with men (MSM). At the end of 2012, the estimated overall prevalence of HIV in Brazil was 0.4% but reached 10.5% among MSM .
Most of the patients in group 1 and almost half of the patients from group 2 had eight years or less of education. Neglected diseases are often found in poor, marginalized sections of the population who have restricted access to formal education . Furthermore, non-white ethnicity/color was prevalent only in group 1. This finding could point to a subgroup with worse social and economic conditions, which historically reflects inequality in access to health services that tends to be secondary to multiple partially overlapping factors, such as social status, gender, race/ethnicity, place of residence, etc. . However, a misreporting of this variable cannot be ruled out because in Brazil, there is a large degree of miscegenation and the registered ethnicity/color was based on skin color instead of proxies of genetic ancestry .
As previously described, HIV clearly modifies the natural history of sporotrichosis and is associated with a broad spectrum of this mycosis . T CD4+ cells have a pivotal role for the control of sporotrichosis  and these cells are exactly the main target of HIV infection. As documented by our findings, HIV co-infection was found to be associated with a much higher incidence of severe disseminated cases and a greater number of hospitalizations and deaths. These patients presented a severe immunosuppression and viral replication. Although further experimental evidence is sorely needed, observational data corroborate the underlying reasoning provided by the pathophysiology and immunology of both infections. This increase in severity may represent a serious issue to the public health and to the economy of the region.
Moreover, we should keep in mind that sporotrichosis is not always a benign disease and can lead to hospitalization and death even in patients without immunosuppression. Since July 2013, sporotrichosis was included among the conditions for which a formal report to the State Health Secretariat is mandatory. This change is an auspicious one, which may contribute to much more accurate reports in the near future.
Secondary bacterial infection and hypersensitivity reactions were found to be relevant causes of hospitalization among patients from group 2. In this group, the presence of comorbidities was key in terms of more serious conditions and more frequent hospitalizations. S. brasiliensis, the main etiologic agent of this specific epidemic, seems to be more virulent than other species of the S. schenckii complex  and may cause pronounced hypersensitivity reactions.
It is remarkable that 47.9% of patients were simultaneously diagnosed with the two infections due to the presence of opportunistic sporotrichosis or other HIV-related conditions. It is clear that this subgroup of patients did not have adequate access to the early diagnosis of HIV infection and have entered into HIV care relatively late, which seems to have increased their chance of acquiring additional infections and the risk of dying as a consequence of AIDS in the first year of diagnosis .
Unlike sporotrichosis, which has been a sustained and protracted threat in recent years, HAART has been associated with a stabilization of the number of classical opportunistic mycoses, such as histoplasmosis and cryptococcosis , as documented in our database.
Sporotrichosis incidence among HIV-infected patients has been increasing on a continuous basis, and at the end of the study period, the incidence was roughly comparable to that of histoplasmosis and cryptococcosis. In contrast, paracoccidioidomycosis does not seem to be associated with HIV infection.
Since the beginning of the sporotrichosis zoonotic epidemic in Rio de Janeiro, IPEC has been the main regional reference center for this mycosis. The same does not apply to the management of classical opportunistic mycoses; therefore, the IPEC figures for sporotrichosis tend to be close to the actual number of cases in the metropolitan area of Rio de Janeiro, but IPEC figures certainly underestimate classical opportunistic mycoses that are usually managed by clinicians and infectious diseases from a large network of primary and secondary HIV/AIDS care locations.
In 2012, the clinical profiles of 21 of these 48 patients of group 1 who were followed up in 1999–2009 were analyzed by our team . A search for international reports in English at that time accounted for 34 cases historically reported. The present study updated this information up to March 2013, making this case series the largest worldwide to the best of our knowledge.
The harms and risks associated with the propagation of sporotrichosis in a disenfranchised population affected by different medical and social conditions are of concern. Among these multiple, partially superimposed burdens, sporotrichosis and HIV co-infection is of great concern. Both infections are preventable and should be targeted by integrated programs. It would be naïve to suppose that these deprived and underserved communities do not face other major overlapping problems, such as substance misuse, crime, and a myriad of other problems associated with inadequate sanitation, waste disposal, access to healthy food, etc. Community development and structural changes fostered by comprehensive public policies and private-public partnerships remain the only real alternative to permit these communities to regain full citizenship and acceptable standards of life. The unabated spread of sporotrichosis in the second largest and most industrialized metropolitan area in Brazil, for more than a decade, is evidence that we are unfortunately far from reaching these goals.
Thanks to the staff of the Laboratory of Mycology, IPEC/Fiocruz.
Conceived and designed the experiments: DFSF ACFdV RVdS RMZO FIB MCGG. Performed the experiments: DFSF ACFdV RMZO MCGG. Analyzed the data: DFSF MBTdS DPC FIB MCGG. Contributed reagents/materials/analysis tools: ACFdV MBTdS DPC MRL RVdS VGV RMZO. Wrote the paper: DFSF DPC VGV FIB MCGG. Patients' follow-up: DFSF ACFdV MRL RVdS VGV MCGG.
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