New Immunological Markers in Chromoblastomycosis-The Importance of PD-1 and PD-L1 Molecules in Human Infection.

The pathogenesis of chromoblastomycosis (CBM) is associated with Th2 and/or T regulatory immune responses, while resistance is associated with a Th1 response. However, even in the presence of IFN-γ, fungi persist in the lesions, and the reason for this persistence is unknown. To clarify the factors associated with pathogenesis, this study aimed to determine the polarization of the cellular immune response and the densities of cells that express markers of exhaustion in the skin of CBM patients. In the skin of patients with CBM, a moderate inflammatory infiltrate was observed, characterized primarily by the occurrence of histiocytes. Analysis of fungal density allowed us to divide patients into groups that exhibited low and high fungal densities; however, the intensity of the inflammatory response was not related to mycotic loads. Furthermore, patients with CBM exhibited a significant increase in the number of CD4+ and CD8+ cells associated with a high density of IL-10-, IL-17-, and IFN-γ-producing cells, indicating the presence of a chronic and mixed cellular immune response, which was also independent of fungal load. A significant increase in the number of PD-1+ and PD-L1+ cells was observed, which may be associated with the maintenance of the fungus in the skin and the progression of the disease.

Draft Genome Sequence of the Rhinocladiella similis Clinical Isolate CBS 149759.

Rhinocladiella similis is a melanized fungi involved in chromoblastomycosis. R. similis genome has never been sequenced, therefore we propose the first draft genome of R. similis.

Pediatric Phaeohyphomycosis: A 44-Year Systematic Review of Reported Cases.

BACKGROUND: Phaeohyphomycosis is an infection caused by pigmented fungi, which can be life-threatening in immunocompromised hosts and in disseminated disease. In adults with disseminated disease, mortality is as high as 79%. Data in children are derived from case reports and series. We conducted this study to review the characteristics of phaeohyphomycoses in children. METHODS: We conducted this study following the PRISMA 2020 guideline for reporting systematic reviews. We performed a review of the reported cases of pediatric phaeohyphomycoses in core bibliographic databases published in the English and Spanish language, between June 1977 and October 2021. We included all eligible cases in patients <18 years to determine the clinical characteristics, diagnosis, treatment, and outcomes. RESULTS: A total of 130 cases were reviewed. The mean age was 8 years. The most common underlying conditions and risk factors included hematologic malignancies (32.5%), neutropenia (26.9%), steroid therapy (24.6%), trauma or surgery (23.1%), and children that received a transplant (14.6%). The most common presentation was localized infection (61.5%); skin and soft tissue infections were the most prevalent (25.4%). Exserohilum spp (20.8%) and Exophiala spp (17.7%) were the most common organisms isolated. Antifungal therapy remains as the most frequent treatment (87%). Overall mortality rate was 22.3% (localized 13.7% vs disseminated 37.3%). CONCLUSION: The findings of this review suggest that phaeohyphomycoses in children have a better outcome compared to adults. We report a lower mortality rate in children when compared with adults in disseminated infection (37.3% vs 79%) and CNS infection (50% vs 60-70%). However, there is a wide variation in mortality rates according to the infection site, treatment, and underlying conditions. Prospective studies are needed.

Phaeohyphomycosis in China.

Background: Due to more attentions paid to melanized fungi over the past few decades and under the background of the global coronavirus disease 2019 pandemic (COVID-19) the fact that the virus itself and the immunosuppressive agents such as glucocorticoids can further increase the risk of infections of deep mycoses, the number of patients with phaeohyphomycosis (PHM) has a substantial increase. Their spectrum is broad and the early diagnosis and treatments are extremely sticky. This study aims to more comprehensively understand the clinical features of phaeohyphomycosis in China over 35 years and to establish a more applicable systematical classification and severity grades of lesions to guide treatments and prognosis. Methods: We reviewed 174 cases of proven phaeohyphomycosis reported in Chinese and English language literature from 1987 to 2021 and we also made the accurate classification definitions and detailed information about the epidemiology, species of clinical dematiaceous fungi, minimum inhibitory concentration values, clinical features, treatments, and prognosis. Results: The mortality of cerebral, disseminated and pulmonary phaeohyphomycosis are 55%, 36%, and 25%. Nearly 19% of patients had poor quality of life caused by the complications such as disability, disfigurements, and blindness. The overall misdiagnosis rate of phaeohyphomycosis was 74%. Moderate to severe rashes are accounting for 82% of subcutaneous phaeohyphomycosis. The areas of the head and face are mostly affected accounting for 16% of severe rashes. Nearly 30% of invasive infections of phaeohyphomycosis are triggered by recurrent lesions. Voriconazole, itraconazole, amphotericin B deoxycholate (AmB-DOC), and terbinafine were most commonly used but diagnosis and treatments of phaeohyphomycosis remain challenging in reality. Conclusions: Our classifications are likely to be more practical and easier to popularize, and there are still also plenty of characteristics in these non-specific lesions. There're no significant variations in cure rates, or death rates between three grades of lesions. But patients with severe rashes have longer courses and lower effective rates.

Gram stain to the rescue: a case report of cerebral phaeohyphomycosis by Cladophialophora bantiana in an immunocompetent 24-year-old.

BACKGROUND: Fungal brain abscesses in immunocompetent patients are exceedingly rare. Cladophialophora bantiana is the most common cause of cerebral phaeohyphomycosis, a dematiaceous mold. Radiological presentation can mimic other disease states, with diagnosis through surgical aspiration and growth of melanized fungi in culture. Exposure is often unknown, with delayed presentation and diagnosis. CASE PRESENTATION: We present a case of cerebral phaeohyphomycosis in a 24-year-old with no underlying conditions or risk factors for disease. He developed upper respiratory symptoms, fevers, and headaches over the course of 2 months. On admission, he underwent brain MRI which demonstrated three parietotemporal rim-enhancing lesions. Stereotactic aspiration revealed a dematiaceous mold on staining and the patient was treated with liposomal amphotericin B, 5-flucytosine, and posaconazole prior to culture confirmation. He ultimately required surgical excision of the brain abscesses and prolonged course of antifungal therapy, with clinical improvement. CONCLUSIONS: Culture remains the gold standard for diagnosis of infection. Distinct microbiologic findings can aid in identification and guide antimicrobial therapy. While little guidance exists on treatment, patients have had favorable outcomes with surgery and combination antifungal therapy. In improving awareness, clinicians may accurately diagnose disease and initiate appropriate therapy in a more timely manner.

Black Fungi and Hydrocarbons: An Environmental Survey for Alkylbenzene Assimilation.

Environmental pollution with alkylbenzene hydrocarbons such as toluene is a recurring phenomenon. Their toxicity and harmful effect on people and the environment drive the search for sustainable removal techniques such as bioremediation, which is based on the microbial metabolism of xenobiotic compounds. Melanized fungi present extremophilic characteristics, which allow their survival in inhospitable habitats such as those contaminated with hydrocarbons. Screening methodologies for testing the microbial assimilation of volatile organic compounds (VOC) are scarce despite their importance for the bioremediation of hydrocarbon associated areas. In this study, 200 strains of melanized fungi were isolated from four different hydrocarbon-related environments by using selective methods, and their biodiversity was assessed by molecular and ecological analyses. Seventeen genera and 27 species from three main orders, namely Chaetothyriales, Cladosporiales, and Pleosporales, were identified. The ecological analysis showed a particular species distribution according to their original substrate. The isolated strains were also screened for their toluene assimilation potential using a simple and inexpensive methodology based on miniaturized incubations under controlled atmospheres. The biomass produced by the 200 strains with toluene as the sole carbon source was compared against positive and negative controls, with glucose and with only mineral medium, respectively. Nineteen strains were selected as the most promising for further investigation on the biodegradation of alkylbenzenes.

Dermatophytic melanonychia: A case series of an increasing disease.

BACKGROUND: Melanonychia refers to brown-black colour pigmentation due to melanin or not-melanin deposition in the nail plate. Onychoscopy allows to distinguish if the pigmentation is due by melanin or not. The main causes of non-melanic pigmentation are subungual haematoma and pigmented onychomycosis. Fungal melanonychia (FM) is rare and may present as diffuse or longitudinal pigmentation. Differential diagnosis includes melanic activation, such as ethnic-type nail pigmentation or frictional melanonychia, but also versus melanic proliferation, such as nevus or nail melanoma. Fungal melanonychia can be due to a colonisation by fungi with black variant or by melanin activation due to inflammation of fungal invasion. OBJECTIVES: The aim of paper is to increase clinical and dermoscopic knowledge of this increasingly frequent disease. METHODS: In this retrospective observational study, twenty patients with dermatophytic melanonychia were collected, with available clinical and dermoscopic pictures. The diagnosis of dermatophytic melanonychia was made based on clinical manifestation and mycological examination. KOH smear was performed in all cases. For each patient, clinical data included: age, gender, type of melanonychia and involved fingers. RESULTS: This study aimed to show increased incidence of dermatophytic melanonychia and its correct management. In addition, we reviewed our collected cases and described the clinical and dermoscopic features of dermatophytic melanonychia. CONCLUSIONS: The results of this study showed that physicians should keep in mind the diagnosis of this increasing disease, and that it cannot be performed relying only on clinical grounds. We would like to highlight the importance of tools as KOH examination, culture and dermoscopy.

New molecular marker for phylogenetic reconstruction of black yeast-like fungi (Chaetothyriales) with hypothetical EIF2AK2 kinase gene.

In eukaryotes, phosphorylation of the α-subunit of eIF2 is a mechanism to adjust cellular gene expression profiles in response to specific signals. The eIF2α kinases are a group of serine-threonine kinases that perform important functions in response to infection, proteotoxicity, and nutrient scavenging. The conserved nature of eIF2α kinases among fungi makes them potential evolutionary markers, which may contribute to deeper understanding of taxonomy and evolution. To date, only few studies are available of eIF2α kinases in black yeasts, which are members of Chaetothyriales containing potential agents of a gamut of major human diseases, such as chromoblastomycosis, phaeohyphomycosis and mycetoma. To establish the phylogenetic validity of sequences of eIF2α kinases hypothetical genes, we compared these genes between members of different classes of fungi, including black yeasts and allies, aiming at evaluation of the phylogeny of this group using an alternative molecular marker, compared to standard ribosomal genes. Trees generated with eIF2α kinase sequences of fungi were compared with those generated by ribosomal internal transcribed spacers (ITS rDNA) sequences from the same species. Sequences used were obtained from the protein Non-redundant database of NCBI, were aligned using CLUSTALX v1.8 and alignments were analyzed with RAxML v8.2.9 on the CIPRES Science Gateway portal. The trees generated had similar topologies, demonstrating that eIF2α kinases hypothetical gene sequences present a coherent reflection of evolution among fungi, compared to trees reconstructed by the use of ribosomal sequences. Our preliminary findings with a limited dataset strongly suggest that the evolution of kinases among black yeasts follows a similar path as revealed by ribosomal data, which underlines the validity of current taxonomy of black yeasts and relatives.

Central line associated blood stream infection (CLABSI) due to Exophiala dermatitidis in an adult patient: Case report and review.

Exophiala dermatitidis is a dematiaceous fungus with yeast-like and hyphal growth states that may cause cutaneous and visceral infections. Recently, E. dermatitidis has been linked to central line associated blood stream infection (CLABSI), probably due to its ability to produce extracellular polysaccharides and grow as biofilm. We describe an E. dermatitidis CLASBI. The strain was identified by morphological and molecular methods. E. dermatitidis CLASBI is highly uncommon, but seems to be increasing.

Phaeohyphomycosis caused by Medicopsis romeroi in solid organ transplant recipients: Report of two cases and comprehensive review of the literature.

Medicopsis romeroi is a melanized coelomycetous fungus, mainly found in tropical and subtropical regions and an uncommon cause of infection in solid organ transplant (SOT) recipients. We describe two cases of SOT recipients diagnosed with phaeohyphomycosis due to M romeroi and provide a comprehensive literature review. These infections should be considered in patients native to tropical countries with a localized skin and soft tissue infection. Sequencing is needed for accurate identification of uncommon melanized fungi. Surgical treatment is recommended to cure the infection and co-adjunctive oral antifungals should be considered.

MALDI-TOF MS-Based Identification of Melanized Fungi is Faster and Reliable After the Expansion of In-House Database.

PURPOSE: Invasive fungal infections caused by melanized fungi are a growing concern. Rapid and reliable identification plays an important role in optimizing therapy. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based identification has emerged as a faster and more accurate diagnostic technique. However, lack of a protein extraction protocol and limited database restricts the identification of melanized fungi by MALDI-TOF MS. The study is designed to standardize protein extraction protocol, to enrich the existing, and to create an in-house database for the rapid identification of melanized fungi. EXPERIMENTAL DESIGN: In this study, 59 sequence-confirmed, melanized fungi were used to expand and to create an in-house database using a modified protein extraction protocol. A total of 117 clinical isolates are further used to validate the created database. RESULT: Using existing Bruker database, only 29(24.8%) out of 117 moulds could be identified. However, all the isolates are identified accurately by supplementing the Bruker database with the created in-house database. MALDI-TOF MS takes significantly lesser time for identification compared to DNA sequencing. CONCLUSION AND CLINICAL RELEVANCE: An expanded database with modified protein extraction protocol can reduce significant time to identify melanized fungi by MALDI-TOF MS.

Combination of Amphotericin B and Terbinafine against Melanized Fungi Associated with Chromoblastomycosis.

Our in vitro studies showed that a combination of amphotericin B and terbinafine had synergistic effects against the majority of melanized fungi associated with chromoblastomycosis (CBM) and similar infections, including those with Cladophialophora carrionii, Cladophialophora arxii, Exophialadermatitidis, Exophialaspinifera, Fonsecaea monophora, Fonsecaea nubica, Fonsecaea pedrosoi, and Phialophora verrucosa. This drug combination could provide an option for the treatment of severe or unresponsive cases of CBM, particularly in cases due to species of Fonsecaea and Cladophialophora.

In Vitro Activities of Luliconazole, Lanoconazole, and Efinaconazole Compared with Those of Five Antifungal Drugs against Melanized Fungi and Relatives.

The in vitro activities of novel azoles compared to those of five antifungal drugs against clinical (n = 28) and environmental (n = 102) isolates of black mold and melanized yeast were determined. Luliconazole and lanoconazole had the lowest geometric mean MICs, followed by efinaconazole, against tested isolates compared to the other drugs. Therefore, it appears that these new imidazole and triazole drugs are promising candidates for the treatment of infections due to melanized fungi and their relatives.

Potential for biocontrol of melanized fungi by actinobacteria isolated from intertidal region of Ilha Do Mel, Paraná, Brazil

Abstract Actinobacteria occur in many environments and have the capacity to produce secondary metabolites with antibiotic potential. Identification and taxonomy of actinobacteria that produce antimicrobial substances is essential for the screening of new compounds, and sequencing of the 16S region of ribosomal DNA (rDNA), which is conserved and present in all bacteria, is an important method of identification. Melanized fungi are free-living organisms, which can also be pathogens of clinical importance. This work aimed to evaluate growth inhibition of melanized fungi by actinobacteria and to identify the latter to the species level. In this study, antimicrobial activity of 13 actinobacterial isolates from the genus Streptomyces was evaluated against seven melanized fungi of the genera Exophiala, Cladosporium, and Rhinocladiella. In all tests, all actinobacterial isolates showed inhibitory activity against all isolates of melanized fungi, and only one actinobacterial isolate had less efficient inhibitory activity. The 16S rDNA region of five previously unidentified actinobacterial isolates from Ilha do Mel, Paraná, Brazil, was sequenced; four of the isolates were identified as Streptomyces globisporus subsp. globisporus, and one isolate was identified as Streptomyces aureus. This work highlights the potential of actinobacteria with antifungal activity and their role in the pursuit of novel antimicrobial substances.

Molecular and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry-Based Characterization of Clinically Significant Melanized Fungi in India.

Melanized or black fungi are a heterogeneous group of fungi causing cutaneous to systemic diseases with high mortality. These fungi are rarely reported as agents of human infections, primarily due to difficulties in their classical identification. In this study, we examined, using molecular methods and matrix-assisted laser desorption ioniazation-time of flight mass spectrometry (MALDI-TOF MS), the diversity of melanized fungi (MF) isolated from patients in 19 medical centers in India. Overall, during a 4-year period, 718 (5.3%) clinical specimens yielded MF. Of these, 72 (10%) isolates had clinical significance and were identified primarily by sequencing the internal transcribed spacer and large subunit (LSU) regions. MF represented 21 genera comprising 29 species, the majority of them belonging to the orders Pleosporales (50%) and Chaetothyriales (22%). Among the 29 fungal species identified in this study, only 6 (20%) species were identified by the MALDI-TOF MS due to the limited commercial database of Bruker Daltonics for MF. However, a 100% identification rate of 20 additional species identified in this study was obtained by constructing an in-house database using 24- to 96-h-old liquid cultures. Further, the CLSI broth microdilution method revealed low MICs for posaconazole (≤1 µg/ml) and voriconazole (≤2 µg/ml) in 96% and 95% of isolates, respectively. Skin/subcutaneous and sino-nasal and pulmonary phaeohyphomycosis due to MF were diagnosed in 21.4% (n = 15) and 28.5% (n = 20) of cases. Also, 10% of patients had central nervous system involvement (n = 7), and 3 cases of fungal osteomyelitis due to Cladophialophora bantiana and Corynespora spp. were observed.

Melanized fungal infections in kidney transplant recipients: contributions to optimize clinical management.

OBJECTIVES: This is a retrospective and observational study addressing clinical and therapeutic aspects of melanized fungal infections in kidney transplant recipients. METHODS: We retrospectively reviewed medical records of all patients admitted between January 1996 and December 2013 in a single institution who developed infections by melanized fungi. RESULTS: We reported on 56 patients aged between 30 and 74 years with phaeohyphomycosis or chromoblastomycosis (0.54 cases per 100 kidney transplants). The median time to diagnosis post-transplant was 31.2 months. Thirty-four (60.8%) infections were reported in deceased donor recipients. Fifty-one cases of phaeohyphomycosis were restricted to subcutaneous tissues, followed by two cases with pneumonia and one with brain involvement. Most dermatological lesions were represented by cysts (23/51; 45.1%) or nodules (9/51; 17.9%). Exophiala spp. (34.2%) followed by Alternaria spp. (7.9%) were the most frequent pathogens. Graft loss and death occurred in two patients and one patient, respectively. Regarding episodes of subcutaneous phaeohyphomycosis, a complete surgical excision without antifungal therapy was possible in 21 of 51 (41.2%) patients. Long periods of itraconazole were required to treat the other 30 (58.8%) episodes of subcutaneous disease. All four cases of chromoblastomycosis were treated only with antifungal therapy. CONCLUSIONS: Melanized fungal infections should be considered in the differential diagnosis of all chronic skin lesions in transplant recipients. It is suggested that the impact of these infections on graft function and mortality is low. The reduction in immunosuppression should be limited to severely ill patients.

Potential for biocontrol of melanized fungi by actinobacteria isolated from intertidal region of Ilha Do Mel, Paraná, Brazil.

Actinobacteria occur in many environments and have the capacity to produce secondary metabolites with antibiotic potential. Identification and taxonomy of actinobacteria that produce antimicrobial substances is essential for the screening of new compounds, and sequencing of the 16S region of ribosomal DNA (rDNA), which is conserved and present in all bacteria, is an important method of identification. Melanized fungi are free-living organisms, which can also be pathogens of clinical importance. This work aimed to evaluate growth inhibition of melanized fungi by actinobacteria and to identify the latter to the species level. In this study, antimicrobial activity of 13 actinobacterial isolates from the genus Streptomyces was evaluated against seven melanized fungi of the genera Exophiala, Cladosporium, and Rhinocladiella. In all tests, all actinobacterial isolates showed inhibitory activity against all isolates of melanized fungi, and only one actinobacterial isolate had less efficient inhibitory activity. The 16S rDNA region of five previously unidentified actinobacterial isolates from Ilha do Mel, Paraná, Brazil, was sequenced; four of the isolates were identified as Streptomyces globisporus subsp. globisporus, and one isolate was identified as Streptomyces aureus. This work highlights the potential of actinobacteria with antifungal activity and their role in the pursuit of novel antimicrobial substances.

Chromoblastomycosis.

Chromoblastomycosis (CBM), also known as chromomycosis, is one of the most prevalent implantation fungal infections, being the most common of the gamut of mycoses caused by melanized or brown-pigmented fungi. CBM is mainly a tropical or subtropical disease that may affect individuals with certain risk factors around the world. The following characteristics are associated with this disease: (i) traumatic inoculation by implantation from an environmental source, leading to an initial cutaneous lesion at the inoculation site; (ii) chronic and progressive cutaneous and subcutaneous tissular involvement associated with fibrotic and granulomatous reactions associated with microabscesses and often with tissue proliferation; (iii) a nonprotective T helper type 2 (Th2) immune response with ineffective humoral involvement; and (iv) the presence of muriform (sclerotic) cells embedded in the affected tissue. CBM lesions are clinically polymorphic and are commonly misdiagnosed as various other infectious and noninfectious diseases. In its more severe clinical forms, CBM may cause an incapacity for labor due to fibrotic sequelae and also due to a series of clinical complications, and if not recognized at an early stage, this disease can be refractory to antifungal therapy.

Fungal Keratitis in North India: Spectrum of Agents, Risk Factors and Treatment.

To assess the prevalence of fungal keratitis, we conducted a retrospective study over 7 years (2005 through 2011) at a tertiary care center in North India. Effort has been made to analyze the disease burden, spectrum of agents and treatment history. The findings were compared with an earlier study at the same center for any change in the epidemiology of the disease. Microbiology records were screened at the Postgraduate Institute of Medical Education and Research, Chandigarh, India, to identify fungal keratitis cases, and available clinical records of those cases were analyzed. Of 2459 clinically suspected fungal keratitis cases, 765 (31 %) cases were direct microscopy confirmed. Of these microscopy-confirmed cases, fungi were isolated in 393 (51.4 %), with Aspergillus spp. ranked top (n = 187, 47.6 %), followed by melanized fungi (n = 86, 21.9 %) and Fusarium spp. (n = 64, 16 %). A male predominance of 78.7 % was noted with a peak in the incidence of fungal keratitis during post-monsoon season (September to November). A delay in diagnosis was significantly associated (p < 0.001) with keratitis cases due to melanized fungi. In comparison with an earlier study, higher isolation of melanized fungi was noted with a widening of the spectrum of agents identified. Thus, fungal keratitis due to Aspergillus spp. remains a serious ocular illness among the active male population in North India with relative rise of keratitis due to melanized fungi. The spectrum of agents causing fungal keratitis has broadened with many rare fungi that are implicated.

Squamous cell carcinoma derived from chronic chromoblastomycosis in Brazil.

BACKGROUND: Chromoblastomycosis (CBM) is a chronic fungal infection caused mainly by the melanized fungi Fonsecaea species. The chronic lesions may be predisposed to develop into cancer, the most serious complication of the disease. METHODS: In this report, 7 cases of squamous cell carcinoma (SCC) resulting from chronic CBM in patients from Maranhão in the Brazilian Amazon are described. RESULTS: The 7 patients presented with SCC that resulted from chronic CBM, caused by Fonsecaea species >10 years' duration. The malignant lesions occurred independent of the antifungal therapy and all patients underwent curative amputation, except for 1 patient who developed metastases in the inguinal and intra-abdominal lymph nodes and thigh muscles. A majority of previous reports have focused on the malignant transformation of CBM described in only 1 patient each. This is a first report describing a group of patients from a single Brazilian state. CONCLUSIONS: Here, we provide new epidemiologic data on malignant CBM lesions, an endemic disease that is seemingly neglected worldwide. We reinforce the idea that typically chronic lesions may be predisposed to turn malignant.