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Chromoblastomycosis
review-article
Publication date (Electronic):
22 October 2014
Publisher:
Termedia Publishing House
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Abstract
Chromoblastomycosis is a chronic fungal infection of the skin and subcutaneous tissue. The infection usually results from a traumatic injury and inoculation of microorganism from a specific group of dematiaceous fungi (usually Fonsecaea pedrosoi, Phialophora verrucosa, Cladophialophora carrionii). In the tissues fungi produce characteristic sclerotic cells or muriform cells. Dermal lesions can range from small nodules to large papillary-like eruptions. The disease has been described worldwide but the prevalence is higher in rural populations in countries with a tropical or subtropical climate, such as Madagascar in Africa and Brazil in South America. Diagnostic techniques are based on direct examination, culture and histopathology. Despite a variety of treatment modalities, which include long courses of antifungals, surgical excision and destructive physical therapies, the disease remains one of the most difficult deep mycotic infections to eradicate.
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Most cited references128
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Mycosphaerella is polyphyletic
P.W. Crous, U. Braun, J.Z. Groenewald (2007)
Mycosphaerella, one of the largest genera of ascomycetes, encompasses several thousand species and has anamorphs residing in more than 30 form genera. Although previous phylogenetic studies based on the ITS rDNA locus supported the monophyly of the genus, DNA sequence data derived from the LSU gene distinguish several clades and families in what has hitherto been considered to represent the Mycosphaerellaceae. Several important leaf spotting and extremotolerant species need to be disposed to the genus Teratosphaeria, for which a new family, the Teratosphaeriaceae, is introduced. Other distinct clades represent the Schizothyriaceae, Davidiellaceae, Capnodiaceae, and the Mycosphaerellaceae. Within the two major clades, namely Teratosphaeriaceae and Mycosphaerellaceae, most anamorph genera are polyphyletic, and new anamorph concepts need to be derived to cope with dual nomenclature within the Mycosphaerella complex.
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Chromoblastomycosis: an overview of clinical manifestations, diagnosis and treatment.
G Vitale, Flavio Queiroz-Telles, P Esterre … (2009)
Chromoblastomycosis is one of the most frequent infections caused by melanized fungi. It is a subcutaneous fungal infection, usually an occupational related disease, mainly affecting individuals in tropical and temperate regions. Although several species are etiologic agents, Fonsecaea pedrosoi and Cladophialophora carrionii are prevalent in the endemic areas. Chromoblastomycosis lesions are polymorphic and must be differentiated from those associated with many clinical conditions. Diagnosis is confirmed by the observation of muriform cells in tissue and the isolation and the identification of the causal agent in culture. Chromoblastomycosis still is a therapeutic challenge for clinicians due to the recalcitrant nature of the disease, especially in the severe clinical forms. There are three treatment modalities, i.e., physical treatment, chemotherapy and combination therapy but their success is related to the causative agent, the clinical form and severity of the chromoblastomycosis lesions. There is no treatment of choice for this neglected mycosis, but rather several treatment options. Most of the patients can be treated with itraconazole, terbinafine or a combination of both. It is also important to evaluate the patient's individual tolerance of the drugs and whether the antifungal will be provided for free or purchased, since antifungal therapy must be maintained in long-term regimens. In general, treatment should be guided according to clinical, mycological and histopathological criteria.
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Restoration of Pattern Recognition Receptor Costimulation to Treat Chromoblastomycosis, a Chronic Fungal Infection of the Skin
Introduction Chromoblastomycosis is a chronic nonfatal mycosis involving the skin and subcutaneous tissues, which is caused by a number of melanized fungi. The disease occurs worldwide, but is observed most frequently in tropical and subtropical regions of Africa and Latin America (Ameen, 2009; Santos et al., 2007). Infection is acquired by the accidental inoculation of the etiologic agent into the subcutaneous tissues, but it usually takes decades following inoculation before clinical symptoms develop. The infection is characterized by erythematous papules, which develop with varying morphology, and systemic invasion is rare (Ameen, 2009; Santos et al., 2007). There are no standard treatments, although approaches usually include chemotherapy, multiple surgical excisions, and/or cryosurgery with liquid nitrogen. Furthermore, there is often a poor response to oral antifungal drugs, and most attempts at treatment have only a modest success rate (Ameen, 2009; Santos et al., 2007). A number of melanized dematiaceous fungi have been associated with chromoblastomycosis, but the most common agent causing this disease is Fonsecaea pedrosoi (Bonifaz et al., 2001). Little is known about this fungus, its cell wall structure, how it is recognized by the host, or the protective /nonprotective immune responses that are triggered upon infection. Host defense against experimental chromoblastomycosis has been shown to rely mainly on the ingestion and elimination of fungal cells by cells of the innate immune system, especially neutrophils and macrophages, but there is also some evidence supporting a requirement of CD4+, although not CD8+, T cell-mediated immune responses (Ameen, 2009; Santos et al., 2007). Notable features of patients with chromoblastomycosis include increased IL-10 and low levels of IFN-γ (Ameen, 2009; Santos et al., 2007). We were interested in understanding the reasons underlying the chronic nature of infection with F. pedrosoi and wanted to explore the possibility that the chronicity of this infection might stem from an inappropriate innate immune response. In this report, we show that F. pedrosoi is recognized by C-type lectin receptors (CLRs), but that there is a lack of sufficient costimulation of the Toll-like receptors (TLRs), and that this results in defective inflammatory responses. Excitingly, we could re-establish this costimulatory cytokine response by exogenous administration of TLR agonists, which could also be used to resolve the infection in vivo. Results Establishment and Characterization of a Murine Model of Chromoblastomycosis To explore the reasons underlying the chronicity of chromoblastomycosis, we made use of an established murine model (Cardona-Castro and Agudelo-Flórez, 1999), where mice are infected intraperitoneally (i.p.) with F. pedrosoi conidia, following which the organism disseminates to the liver and spleen, where it persists for many weeks (Figure 1A). By comparison, infection with a pathogen causing an acute infection, such as Candida albicans, is cleared rapidly (Tsoni et al., 2009) (dotted line in Figure 1A). Characterization of the spleens of the infected animals revealed high levels of IL-10 as well as low levels of TNF and IFN-γ, cytokine profiles similar to those described in infected humans (Ameen, 2009; Santos et al., 2007) (Figure 1B). To determine whether IL-10 was contributing to the persistence of the infection in our mice, we compared fungal burdens and cytokine profiles in wild-type and IL-10-deficient animals 7 days after infection. This time point was chosen as it allowed us to examine primarily the innate response to this pathogen (see later). Unexpectedly, the lack of IL-10 only marginally reduced the fungal burdens in the organs of the infected animals and did not greatly influence the inflammatory responses to this pathogen, as determined by measuring the levels of TNF in the infected organs (Figures 1C and S1). Thus, these results suggested that IL-10 was not a major factor involved in establishing the chronicity of infection with F. pedrosoi. F. pedrosoi Fails to Induce Inflammatory Responses from Macrophages We next explored the innate recognition of F. pedrosoi by characterizing its interactions with thioglycollate-elicited macrophages in vitro. We found that the conidia bound to macrophages in a dose-dependent fashion, yet surprisingly failed to stimulate the production of TNF, even at high multiplicities of infection (moi) (Figure 2A). In contrast, recognition of the fungal particle zymosan, which was included as a control in these experiments, induced a robust inflammatory response, as expected (Brown et al., 2003). To explore the possibility that F. pedrosoi was actively suppressing leukocyte inflammatory responses, we examined the interaction of macrophages with heat-killed (HK) conidia. However, despite the heat treatment, macrophages still failed to respond to the conidia (Figure 2B). In addition, prolonged coculture for 20 hr with viable conidia, during which time the pathogen formed hyphae, similarly did not result in the induction of TNF (data not shown). The targeting of complement receptor 3 (CR3) is another suppressive mechanism of fungi (Brandhorst et al., 2004); however, loss of CR3 did not restore TNF responses to F. pedrosoi conidia (Figure 2C). Thus, the lack of inflammatory responses to F. pedrosoi was due to a failure of innate recognition and not active suppression by the pathogen. Inflammatory Responses to F. pedrosoi Can Be Reinstated by TLR Costimulation We have previously demonstrated that the recognition of fungal β-glucans by macrophages is not sufficient to induce inflammatory responses, in the absence of additional costimulation through MyD88-coupled TLRs (Brown et al., 2003; Dennehy et al., 2008; Rosas et al., 2008). Thus, we next considered the possibility that the failure of F. pedrosoi conidia to induce inflammatory responses was due to a lack of PRR costimulation. We first determined whether β-glucans were exposed in F. pedrosoi and could demonstrate that both conidial and hyphal forms of this pathogen displayed these carbohydrates at the cell surface (Figure 2D). Interestingly, heat killing did not significantly alter the level of β-glucan exposure (Figure S2A). Using transfected macrophages, we could also demonstrate that the β-glucan receptor Dectin-1 could directly mediate the binding of F. pedrosoi conidia to host cells (Figure 2E). We then explored the effect of TLR costimulation on macrophage inflammatory responses by making use of substimulatory doses of the TLR2 agonist Pam3CSK4, which we had previously shown to work synergistically with Dectin-1 (Dennehy et al., 2008). Remarkably, while the addition of F. pedrosoi conidia to macrophages failed to induce significant levels of TNF, as we had observed before, costimulation with Pam3CSK4 induced robust responses (Figure 2F). Similar results were also observed using human peripheral blood-derived macrophages and murine bone-marrow-derived dendritic cells (BMDCs) (Figure 2G). Furthermore, as we had previously demonstrated with purified β-glucan (Dennehy et al., 2008), costimulation with F. pedrosoi could be achieved using multiple TLR agonists, including LPS (TLR4) and Imiquimod (TLR7) (Figures 2G and S2B). It is notable that the stimulation of BMDCs with F. pedrosoi alone induced some TNF, which was expected, as stimulation of the Dectin-1/Syk signaling pathway in dendritic cells (DCs) is known to be sufficient for cytokine induction (Dennehy et al., 2009; Rogers et al., 2005; Rosas et al., 2008).Thus, the inability of F. pedrosoi to induce robust inflammatory responses was due to a lack of costimulation of the TLR pathway. To confirm the role of Dectin-1 in these responses, we next assessed the costimulatory response in Dectin-1-deficient macrophages. As shown previously (Dennehy et al., 2008), the costimulatory response to highly purified β-glucans was ablated in Dectin-1−/− cells. Unexpectedly, Dectin-1-deficient macrophages displayed no alterations in their ability to induce costimulatory responses to F. pedrosoi conidia (Figure 2F). Furthermore, we found that loss of Dectin-1 had no effect on the ability of primary macrophages to bind F. pedrosoi conidia (Figure S2C), and characterization of Dectin-1−/− mice revealed only marginal effects on fungal burdens in the organs of the infected animals (Figure S2D). Thus, Dectin-1 does not contribute to the costimulatory responses that were observed in vitro and plays only a minor role during infection in vivo. F. pedrosoi-TLR Costimulation Requires Mincle and Signaling through the Syk/CARD9 Pathway To determine the signaling pathways and receptors involved in mediating the costimulatory response, we next characterized BMDCs deficient in various signaling molecules. We first examined BMDCs deficient in MyD88 to confirm the involvement of this pathway and, as expected, found that the coaddition of F. pedrosoi and Pam3CSK4 failed to induce a robust inflammatory response in these cells (Figure 3A). Interestingly, the response to F. pedrosoi alone was also partly attenuated, indicating some involvement of the MyD88 pathway in sensing of this pathogen by DCs. Indeed when examined further, we found that mice deficient in MyD88 had enhanced fungal burdens during infection (Figure S3A). We next determined if the costimulatory response required signaling through Syk and CARD9, as we had shown for Dectin-1 (Dennehy et al., 2008), and observed that BMDCs deficient in Syk (Figure 3B) or CARD9 (Figure 3C) failed to induce robust inflammatory responses in the presence of F. pedrosoi and Pam3CSK4. The responses to F. pedrosoi alone were also attenuated in these cells. To date, the only other Syk-coupled receptors that have been implicated in fungal recognition are the CLRs Mincle and Dectin-2, and both signal through the FcRγ chain (Drummond et al., 2011). Hence, we examined the role of this signaling adaptor and found that the costimulatory response was lost in Fcγ−/− BMDCs (Figure 3D). We then explored the possibility of Dectin-2 involvement, by inhibiting this receptor with blocking monoclonal antibodies, but observed no effect on these responses (Figure S3B). In contrast, we found that the costimulatory responses were completely ablated in Mincle−/− BMDCs and that this defect was specific for the recognition of F. pedrosoi, as Mincle−/− BMDCs retained normal responses to LPS (Figures 3E and S3C). Thus, these results identify the Fcγ-coupled CLR Mincle as a major receptor involved in the innate recognition of F. pedrosoi. A model of the proposed costimulatory pathway is shown in Figure 3F. TLR Costimulation Cures F. pedrosoi Infection The failure of F. pedrosoi to induce robust inflammatory responses provides a possible explanation for the persistence of this pathogen, so we investigated the possibility that artificial costimulation of the TLR pathway, to reinstate inflammatory responses, would help resolve the infection in vivo. We first determined if this was possible by administering F. pedrosoi i.p., with or without LPS, and monitoring TNF production in the peritoneal cavity after 3 hr. LPS was chosen for these and subsequent experiments, as the effects and dosage of this TLR agonist in vivo are well characterized and as we had shown it to be able to costimulate inflammatory responses to F. pedrosoi in vitro (Dennehy et al., 2008) (see Figures 2G and S2B). As shown in Figure 4A, the administration of either F. pedrosoi conidia or a low dose of LPS induced little TNF, but when added in combination, they induced a robust inflammatory response. To demonstrate an effect on fungal clearance, mice were infected with F. pedrosoi and the disease was allowed to establish for 3 days, following which a single low dose of LPS was administered either i.p. or i.v., and the infection in the organs was assessed after a further 4 days (Figure 4B). Remarkably, the administration of a single low dose of LPS, via either route, resulted in a near complete clearance of the pathogen from both the spleen and liver, in comparison to the untreated animals (Figures 4C and S4A). Furthermore, as predicted from our in vitro analyses, the administration of LPS significantly increased the levels of TNF in the infected organs (Figures 4C and S4A). This induction of TNF was critical for fungal clearance, since we found LPS to have no beneficial effect in mice deficient in this cytokine (Figures 4D and S4B). In addition, similar effects of LPS on fungal burdens were observed in RAG2−/− mice (Figures 4E and S4C). Our results suggest that the administration of TLR agonists may be a form of treatment for chromoblastomycosis in humans, ideally through the topical application of these agonists to infected skin. However, it is possible that TLR agonists would not be effective in treating subcutaneous infections. To explore this, we infected mice subcutaneously with F. pedrosoi and then treated some animals with topical applications of the FDA-approved agonist Imiquimod, which was also capable of inducing costimulatory activity in vitro (see Figure 2). Remarkably, we found that the topical application of Imiquimod significantly reduced fungal burdens in the skin at day 7 postinfection (Figure 4F). Similar reductions in fungal burdens were also obtained in these tissues following the i.p. administration of LPS on day 3 postinfection. Thus, we conclude that reinstating innate inflammatory responses to F. pedrosoi by artificial TLR costimulation in vivo can help resolve this normally persistent infection. Discussion Very little is known about the immunology underlying chromoblastomycosis or the reasons for the chronicity of the disease. In this report we demonstrate that the chronic nature of this infection stems from inadequate innate recognition and the subsequent failure to mount protective inflammatory responses. This failure was not due to active suppression by F. pedrosoi, as inflammatory responses to HK organisms were similarly defective. Furthermore, this chronicity was not due to high levels of IL-10, as the level of infection was not greatly altered in mice deficient in this immunosuppressive cytokine. Although F. pedrosoi was recognized by leukocyte CLRs, particularly Mincle, we found that this recognition was not sufficient in and of itself to trigger protective inflammatory responses. These findings were reminiscent of β-glucan recognition by Dectin-1, which required costimulation of MyD88-coupled TLRs to induce robust inflammatory responses (Dennehy et al., 2008). Indeed, we found that costimulation of leukocytes with purified TLR agonists induced robust inflammatory responses to F. pedrosoi, indicating that it was the lack of recognition by this PRR family that was responsible for the defective innate responses. Like Dectin-1, the costimulatory response required signaling via the Syk-CARD9 pathway, but was triggered by the FcRγ-coupled CLR Mincle. We could demonstrate this definitively by showing that the costimulatory responses to this organism were lost in cells deficient in any of these signaling components. While it is likely that Mincle is recognizing a mannose-based cell wall component (Yamasaki et al., 2009), the identity of the ligand is unclear. It should also be noted that unlike macrophages, the recognition of F. pedrosoi did induce some response in DCs, even in the absence of TLR costimulation. For other Syk-dependent responses, such as those triggered through Dectin-1, these cellular differences have been linked to the effects of cytokines, such as the GM-CSF used to generate the DCs and the differential usage of CARD9, but the underlying reasons are still not fully understood (Drummond et al., 2011). Nonetheless, as for Dectin-1, TLR costimulation induced robust inflammatory responses to F. pedrosoi in both cell types. The immunostimulatory components within the cell walls of many fungi are thought to be shielded, possibly providing an explanation for the failure of F. pedrosoi to induce protective responses. However, the lack of response to dead organisms indicates that this is unlikely, as heat killing is thought to disrupt the cell wall architecture and expose underlying PAMPs (Robinson et al., 2009). Although this suggests that F. pedrosoi lacks sufficient levels of exposed TLR ligands to stimulate robust responses, the partial ablation of TNF production in the MyD88−/− DCs does indicate the presence of some ligands for these receptors. What the nature of these ligands may be is unclear, as the architecture and composition of the cell wall of F. pedrosoi is largely unknown. Furthermore, the significant increase in fungal burdens in the MyD88-deficient mice might also suggest some contribution of TLR recognition to the control of this pathogen, but the interpretation of these results are complicated by the role of MyD88 in IL-1 receptor signaling and the importance of IL-1 signaling in antifungal immunity. The failure of leukocytes to induce robust inflammatory responses to F. pedrosoi led us to test the possibility that inducing these responses, through the exogenous administration of TLR agonists, could help resolve the infection in vivo. Indeed, using a murine model of systemic infection, we found that the administration of LPS, either i.v. or i.p., significantly reduced fungal burdens in infected organs. This increased fungal clearance was due to the enhanced inflammatory responses triggered by the exogenous costimulation of the TLRs, as LPS failed to have an effect in TNF−/− mice. However, LPS has also been shown to induce Mincle expression in vitro (Matsumoto et al., 1999), therefore raising the possibility of an indirect contribution of this TLR agonist in host responses. While the systemic mouse model of chromoblastomycosis is not an accurate representation of the human subcutaneous infection, it is thought to be the best model for studying the chronic nature of this disease (Cardona-Castro and Agudelo-Flórez, 1999). Indeed, the systemic infection is chronic in mice, and the pathogen persists for many weeks in the organs of untreated animals. Importantly, the ability of artificial TLR costimulation to restore inflammatory responses and induce rapid fungal clearance, particularly in RAG-deficient mice, clearly demonstrates that the persistence of this fungal pathogen is primarily due to defective innate recognition. However, chronic infection with F. pedrosoi also leads to dysregulated adaptive immunity (Mazo Fávero Gimenes et al., 2005), which was not addressed here, and it would be interesting to examine the effect of TLR agonist treatment on the development of these responses during the infection. Excitingly, these results suggest that the exogenous administration of TLR agonists could be used to treat human patients. Although the majority of our studies were performed using disseminated infections, we could demonstrate that a similar approach was also feasible in a subcutaneous infection model. While this approach requires further optimization, we found that the topical application of Imiquimod significantly reduced fungal burdens in the infected tissues. Our in vitro data also suggest that such an approach may work in humans, as human macrophages, like their mouse counterparts, failed to induce robust inflammatory responses to F. pedrosoi in the absence of artificial TLR costimulation. In conclusion, we have shown that the persistence of infection with F. pedrosoi is due to a failure in innate recognition, stemming from a lack of TLR costimulation. It is tempting to speculate that defective PRR costimulation may underlie the development of other chronic infections and that enhancement of inflammatory responses may be responsible for the anti-infective activities reported for many immunostimulants, such as β-glucan. Our results also highlight the importance of coordinated PRR signaling and demonstrate how exogenously applied PRR agonists could be used to treat these types of diseases. For chromoblastomycosis in humans, this might simply involve the topical application of appropriate TLR ligands, such as Imiquimod. Experimental Procedures Animals Male or female 8- to 14-week-old 129Sv, 129Sv Dectin-1−/− (Taylor et al., 2007), BALB/C, BALB/C IL-10−/− (Dewals et al., 2010), C57BL/6, C57BL/6 RAG2−/− (Dewals et al., 2010), C57BL/6 TNF−/− (Marino et al., 1997), and C57BL/6 Myd88−/− (Fremond et al., 2004) mice were obtained from the specific pathogen-free facility of the University of Cape Town. All animal experimentation was performed using groups of 5–10 animals, repeated at least once, and conformed to institutional guidelines for animal care and welfare. F. pedrosoi Growth Conditions and Fluorescent Labeling F. pedrosoi ATCC 46428 was streaked onto potato dextrose agar or Sabouraud agar plates for isolation of individual colonies for 12 days. Colonies were cultured in a shaking incubator for 72 hr at 30°C in potato broth for in vitro and in vivo assays. The conidia were filtered to remove hyphae and washed with PBS before use (live conidia) or were heat killed by boiling for 30 min. For fluorescence labeling, washed live or HK conidia were labeled with Rhodamine Green-X (Invitrogen) (200 μg/ml) for 30 min at 25°C, followed by extensive washing. To detect surface-exposed β-glucans, washed live or HK F. pedrosoi cells were stained with soluble Fc-Dectin-1(Graham et al., 2006) or Fc-CLEC9A (Huysamen et al., 2008) chimeric proteins (5 μg/ml), as described previously (Graham et al., 2006). In some experiments, soluble β-glucan (100 μg/ml) was mixed with Fc-Dectin-1 for 30 min prior to staining. Cells and In Vitro Fungal Stimulations DCs were generated from Syk−/− fetal livers, as described (Rogers et al., 2005), or from the bone marrow of CARD9−/− (Gross et al., 2009), FcRγ−/− (Takai et al., 1994), Mincle−/− (Yamasaki et al., 2009), MyD88, and C57BL/6 mice, using standard protocols. Human monocyte-derived macrophages and thioglycollate-elicited macrophages were generated as described previously (Dennehy et al., 2008; Willment et al., 2003). Macrophages and BMDCs were plated the night before use in 24-well plates at a density of 2.5 × 105 cells per well in RPMI medium with 10% heat-inactivated FCS. The RAW264.7 macrophages expressing Dectin-1 (Brown et al., 2002) and control cells were plated at 2.5 × 105 cells per well in medium containing 0.4 mg/ml G418 (Invitrogen). For the in vitro binding and cytokine assays, unlabeled or Rhodamine Green-X-labeled live or HK F. pedrosoi were added to the cells, as indicated, and incubated for 30 min at 37°C. In some experiments, the following compounds were also added alone or in combination, as indicated: unlabeled or FITC-labeled zymosan (25 particles per cell) (Invitrogen), purified β-glucan particles (100 μg/ml) (Dennehy et al., 2008), Pam3CSK4 (10 ng/ml) (Invivogen), LPS (1 ng/ml) (Sigma), and Imiquimod (1 μg/ml) (Invivogen). Unbound particles were removed by washing. The medium was replaced, and the cells were cultured for a further 3 hr for analysis of TNF by ELISA (BD Biosciences). Cytokine stimulations were not influenced by the presence or absence of a fluorescent label on the fungal particles (data not shown). After incubation, supernatants were stored at −80°C until use, cells were lysed in 3% (volume/volume) Triton X-100, and fluorescence was measured with a Titertek Fluoroskan II (Labsystems). In Vivo Models For in vivo infections, mice were infected with 2 × 106 conidia of F. pedrosoi i.p. In some experiments, LPS (10 ng) (Sigma) was also administered to mice i.p. or i.v., 3 days after infection with F. pedrosoi. At appropriate time points after infection, as indicated in the text, the animals were sacrificed, and colony-forming units were determined in disaggregated whole livers and spleens by serial dilution onto Sabouraud agar plates. Organ cytokine levels were determined by ELISA (BD Biosciences). To measure peritoneal inflammation, mice were injected i.p. with 2 × 106 live F. pedrosoi and/or LPS (10 ng/ml) and were killed after 3 hr. TNF was measured in peritoneal lavage fluid by ELISA (BD Biosciences). For infection of the footpad, mice were subcutaneously injected with 1 × 107 F. pedrosoi conidia, and then 5% Imiquimod cream (Aldara, Graceway Phamaceuticals) was topically applied daily to the site of infection, where indicated. In other mice, 100 ng of LPS was administered i.p. at day 3 after infection. All mice were sacrificed at day 7, and colony-forming units were determined in disaggregated footpads by serial dilution onto Sabouraud agar plates. Statistics Student's t test was used for the analysis of two groups. Results were considered statistically significant with p values of ≤ 0.05.
Author and article information
Journal
Journal ID (nlm-ta): Postepy Dermatol Alergol
Journal ID (iso-abbrev): Postepy Dermatol Alergol
Journal ID (publisher-id): PDIA
Title:
Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii
Publisher:
Termedia Publishing House
ISSN
(Print):
1642-395X
ISSN
(Electronic):
2299-0046
Publication date
(Electronic):
22
October
2014
Publication date
(Print):
October
2014
Volume: 31
Issue: 5
Pages: 310-321
Affiliations
[1
]Department of Mycology, Chair of Microbiology, Jagiellonian University Medical College,
Krakow, Poland. Head of Department: Paweł M. Krzyściak MD, PhD
Author notes
Address for correspondence: Paweł Krzyściak, 18 Czysta St, 31-121 Krakow, Poland. e-mail:
pawel.krzysciak@
123456uj.edu.pl
Article
Publisher ID: 22355DOI: 10.5114/pdia.2014.40949
PMC ID: 4221348
PubMed ID: 25395928
SO-VID: 4554de50-5d89-4b55-a289-169d85eafad5
Copyright © Copyright © 2014 Termedia
License:
This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License, permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
History
Date
received
: 29
October
2013
Date
revision received
: 12
December
2013
Date
accepted
: 19
January
2014
Categories
Subject:
Review Paper
Data availability: