Galleria mellonella in vitro model for chromoblastomycosis shows large differences in virulence between isolates.

BACKGROUND: Chromoblastomycosis is the World Health Organization (WHO)-recognized fungal implantation disease that eventually leads to severe mutilation. Cladophialophora carrionii (C. carrionii) is one of the agents. However, the pathogenesis of C. carrionii is not fully investigated yet. METHODS: We investigated the pathogenic potential of the fungus in a Galleria mellonella (G. mellonella) larvae infection model. Six strains of C. carrionii, and three of its environmental relative C. yegresii were tested. The G. mellonella model was also applied to determine antifungal efficacy of amphotericin B, itraconazole, voriconazole, posaconazole, and terbinafine. RESULTS: All strains were able to infect the larvae, but virulence potentials were strain-specific and showed no correlation with clinical background of the respective isolate. Survival of larvae also varied with infection dose, and with growth speed and melanization of the fungus. Posaconazole and voriconazole exhibited best activity against Cladophialophora, followed by itraconazole and terbinafine, while limited efficacy was seen for amphotericin B. CONCLUSION: Infection behavior deviates significantly between strains. In vitro antifungal susceptibility of tested strains only partly explained the limited treatment efficacy in vivo.

Antifungal Resistance in Pulmonary Aspergillosis.

Aspergilli may cause various pulmonary diseases in humans, including allergic bronchopulmonary aspergillosis (ABPA), chronic pulmonary aspergillosis (CPA), and acute invasive pulmonary aspergillosis (IPA). In addition, chronic colonization may occur in cystic fibrosis (CF). Aspergillus fumigatus represents the main pathogen, which may employ different morphotypes, for example, conidia, hyphal growth, and asexual sporulation, in the various Aspergillus diseases. These morphotypes determine the ease by which A. fumigatus can adapt to stress by antifungal drug exposure, usually resulting in one or more resistance mutations. Key factors that enable the emergence of resistance include genetic variation and selection. The ability to create genetic variation depends on the reproduction mode, including, sexual, parasexual, and asexual, and the population size. These reproduction cycles may take place in the host and/or in the environment, usually when specific conditions are present. Environmental resistance is commonly characterized by tandem repeat (TR)-mediated mutations, while in-host resistance selection results in single-resistance mutations. Reported cases from the literature indicate that environmental resistance mutations are almost exclusively present in patients with IA indicating that the risk for in-host resistance selection is very low. In aspergilloma, single-point mutations are the dominant resistance genotype, while in other chronic Aspergillus diseases, for example, ABPA, CPA, and CF, both TR-mediated and single-resistance mutations are reported. Insights into the pathogenesis of resistance selection in various Aspergillus diseases may help to improve diagnostic and therapeutic strategies.

Comparative analysis of whole genomes and transcriptomes of Microsporum canis from invasive dermatophytosis and tinea capitis.

ABSTRACTGenomes of strains of the zoophilic dermatophyte Microsporum canis from invasive (disseminated and subcutaneous) and noninvasive (tinea capitis) infections were compared. Especially the disseminated strain showed significant syntenic rearrangements, including multiple translocations and inversions, and numerous SNPs and Indels in comparison to the noninvasive strain. In transcriptome analysis, both invasive strains were enriched for GO pathways related to components of the membrane, iron binding and heme binding, which possibly enables them to invade deeper into dermis and blood vessels. At 37 °C, invasive strains showed gene expression enriched for DNA replication, mismatch repair, N-glycan biosynthesis and ribosome biogenesis. The invasive strains were slightly less susceptible to multiple antifungal agents suggesting that acquired elevated drug resistance might be involved in the refractory disease courses. Patient with disseminated infection failed to respond to a combined antifungal treatment with itraconazole, terbinafine, fluconazole and posaconazole.

Asymptomatic Candida glabrata urinary tract infection in an immunocompetent young female: A case report.

INTRODUCTION: Fungal urinary tract infections (UTIs) are becoming increasingly common in hospitalized patients and Candida species are the most prevalent organisms. However, recurrent candiduria in young healthy outpatients is rare thus require further examination to find the etiologic factors. CASE PRESENTATION: We described a case of recurrent asymptomatic c caused by azole-resistant C. glabrata in a healthy young female who only had previous use of antibiotics without other risk factors. However, after removal of the predisposing factor and the use of sensitive antifungal agents, the patient's urine cultures remained positive. This phenomenon indicated to us that the patient might have an immune-related genetic deficiency. We found a novel caspase-associated recruitment domain-containing protein 9 (CARD9) gene mutation (c.808-11G > T) which might be the cause of recurrent asymptomatic candiduria in this immune-competent young female without any underlying diseases. CONCLUSIONS: We report a case of recurrent asymptomatic candiduria caused by azole-resistant Candida glabrata in a young healthy female with a novel CARD9 mutation. A functional study of this mutation should be performed in the future to determine its effect on asymptomatic fungal UTIs.

The first suspected disseminated Hormographiella aspergillata infection in China, diagnosed using metagenomic next-generation sequencing: a case report and literature review.

Hormographiella aspergillata is a rare and emerging cause of invasive mould infections in patients with haematological malignancies, with a mortality rate of approximately 70%. Here, we present the first reported case of suspected disseminated H. aspergillata infection in China. The patient experienced a second relapse of acute myeloid leukaemia and developed neutropenia, fever, discrepant blood pressure between limbs, and cutaneous lesions limited to the left upper extremity. Since lung tissue biopsy was not feasible, metagenomic next-generation sequencing (mNGS) and panfungal polymerase chain reaction (PCR) analysis of bronchoalveolar lavage fluid and blood samples were performed, which indicated probable H. aspergillata pulmonary infection. Histopathology of cutaneous lesions revealed numerous fungal hyphae within dermal blood vessels. mNGS of a skin biopsy sample identified H. aspergillata sequences, and the fungi was subsequently recovered from fungal culture, proving cutaneous H. aspergillata infection. Despite combined antifungal therapy, the patient died owing to disease progression. Additionally, 22 previously reported cases of invasive H. aspergillata infection were reviewed in patients with haematological malignancies. Thus, mNGS is a powerful diagnostic tool for the early and effective detection of invasive H. aspergillata infections, with the advantage of sequencing all potential pathogens, and providing results within 24 h.

Genome sequencing of Cladophialophora exuberans, a novel candidate for bioremediation of hydrocarbon and heavy metal polluted habitats.

Cladophialophora exuberans is a filamentous fungus related to black yeasts in the order Chaetothyriales. These melanized fungi are known for their 'dual ecology', often occurring in toxic environments and also being frequently involved in human infection. Particularly Cladophialophora exuberans, C. immunda, C. psammophila, and Exophiala mesophila have been described with a pronounced ability to degrade aromatic compounds and xenobiotic volatiles, such as benzene, toluene, ethyl-benzene, and xylene, and are candidates for bioremediation applications. The objective of the present study is the sequencing, assembly, and description of the whole genome of C. exuberans focusing on genes and pathways related to carbon and toxin management, assessing the tolerance and bioremediation of lead and copper, and verifying the presence of genes for metal homeostasis. Genomic evaluations were carried out through a comparison with sibling species including clinical and environmental strains. Tolerance of metals was evaluated via a microdilution method establishing minimum inhibitory (MIC) and fungicidal concentrations (MFC), and agar diffusion assays. Heavy metal bioremediation was evaluated via graphite furnace atomic absorption spectroscopy (GFAAS). The final assembly of C. exuberans comprised 661 contigs, with genome size of 38.10 Mb, coverage of 89.9X and a GC content of 50.8%. In addition, inhibition of growth was shown at concentrations of 1250 ppm for copper and at 625 ppm for lead, using the MIC method. In the agar tests, the strain grew at 2500 ppm of copper and lead. In GFAAS tests, uptake capacities were observed of 89.2% and 95.7% for copper and lead, respectively, after 21 experimental days. This study enabled the annotation of genes involved in heavy metal homeostasis and also contributed to a better understanding of the mechanisms used in tolerance of and adaptation to extreme conditions.

Fatal dermatophytic pseudomycetoma in a patient with non-HIV CD4 lymphocytopenia.

Dermatophytic pseudomycetoma is a rare invasive infection, involving both immunocompetent and immunocompromised individuals. Since the discovery of inherited immune disorders such as the impairment of CARD9 gene, extended dermatophyte infections are mostly ascribed to any of these host factors. This study is to present and explore the potential causes in a fatal dermatophytic pseudomycetoma patient. We present a chronic and deep pseudomycetoma caused by the common dermatophyte Microsporum canis which ultimately led to the death of the patient. Mycological examination, genetic studies and host immune responses against fungi were performed to explore the potential factors. The patient had decreased lymphocyte counts with significantly reduced CD4+ T cells, although all currently known genetic parameters proved to be normal. Through functional studies, we demonstrated that peripheral blood mononuclear cells from the patient showed severe impairment of adaptive cytokine production upon fungus-specific stimulation, whereas innate immune responses were partially defective. This is, to our knowledge, the first report of fatal dermatophytic pseudomycetoma in a patient with non-HIV CD4 lymphocytopenia, which highlights the importance of screening for immune deficiencies in patients with deep dermatophytosis.

Challenges towards management of CARD9-deficient patients with phaeohyphomycosis: A case report and case series study.

BACKGROUND: A number of recalcitrant phaeohyphomycosis cases with a life-threatening prognosis have been observed in CARD9-deficient patients, but little is known about the long-term management strategies that are effective for such intractable individuals. OBJECTIVES: To study the genetic and immunological mechanisms underlying recalcitrant phaeohyphomycosis and to share our clinical experiences regarding its treatment. PATIENTS/METHODS: Ten CARD9-deficient patients with recalcitrant phaeohyphomycosis admitted to our centre in the past two decades were followed-up, and their clinical presentations, laboratory findings, treatment and prognoses were analysed; one of them was a novel case of recalcitrant phaeohyphomycosis harbouring CARD9 mutations. Innate and adaptive immunological responses of patient-derived peripheral blood mononuclear cells were evaluated using ELISA and flow cytometry. RESULTS: We identified a total of seven CARD9 mutations in the ten analysed patients. Moreover, patient-derived cells exhibited a significant impairment of innate and adaptive immune responses upon fungus-specific stimulation. All the patients experienced recurrence and exacerbation; four of them died, two exhibited continued disease progress with unsatisfactory therapeutic efficacy, three showed obvious improvement under maintenance therapy, and only one achieved a clinical cure. CONCLUSIONS: Our study highlighted that otherwise healthy patients diagnosed with early-onset, unexplained and recalcitrant phaeohyphomycosis should be analysed for CARD9 mutations and immune deficiency. Thereafter, the length and choice of management remain challengeable and must be adjusted based on the clinical presentations and responses of patients over their lifetimes. Although continued posaconazole treatment may be the promising first-line therapy at present, novel strategies are worth exploring.

Dual RNA-Sequencing and Liquid Chromatography-Mass Spectrometry Unveil Specific Insights on the Pathogenicity of Trichophyton mentagrophytes Complex.

Trichophyton mentagrophytes is increasingly considered to be a public health hazard because it causes the most severe manifestations of dermatophytosis. In this study, we performed a series of studies to determine the pathogenicity of the T. mentagrophytes complex. We show that the T. mentagrophytes complex interacts with keratinocytes through pattern-recognition receptors‒MAPK/noncanonical NF-κB pathways and that the hyphal form of T. mentagrophytes is responsible for the increased inflammatory responses in keratinocytes. Moreover, SN-38 is likely a toxin of T. mentagrophytes that induces apoptosis in keratinocytes both in vivo and in vitro. Our results explain the severe pathogenicity and destructiveness of T. mentagrophytes observed in the clinic and pave the way for designing novel toxin-directed therapies to improve patient outcomes.

Successful treatment of disseminated amphotericin-resistant fusariosis in a paediatric patient with acute lymphoblastic leukaemia: a case report and literature review

Background: Disseminated fusariosis is a rare and fatal infection in immunocompromised patients. Objectives: We report a case of disseminated amphotericin-resistant fusariosis in a paediatric patient with acute lymphoblastic leukaemia and review the features of reported disseminated fusariosis in China. Materials & Methods: Case reports of disseminated fusariosis were searched from the Chinese literature over the last two decades. Results: The presented case is a 15-year-old female who developed fever and multiple painful purple plaques with black necrotic centres and blood blisters. Fusarium was detected in blood and skin lesions with a high minimum inhibitory concentration (MIC) of amphotericin B (AMB) (>32 µg/mL) and a low MIC of voriconazole (VRC) (0.25 µg/mL). The Fusarium fujikuroi species complex was finally identified by rRNA gene analysis. Combination therapy of VRC and terbinafine (TRF) successfully resolved the disease after more than four months of treatment. Based on the review, the most common manifestations of disseminated fusariosis were fever, skin lesions and positive blood cultures, comprising nine cases (64.3%). Other sites of infection, including the lungs, eyes, sinuses or bone marrow, occurred in eight cases (57.1%). Seven patients (50%) were cured after monotherapy or combination therapy with AMB and VRC. Conclusion: In view of this case and the review of the literature, early identification of Fusarium infection and the appropriate antifungal drugs are critical for successful treatment. Primary therapy should consist of VRC or liposomal amphotericin B (L-AMB), with salvage therapy consisting of posaconazole (PSC). The combination of antifungals is probably necessary and more effective.

Ten decadal advances in fungal biology leading towards human well-being.

Fungi are an understudied resource possessing huge potential for developing products that can greatly improve human well-being. In the current paper, we highlight some important discoveries and developments in applied mycology and interdisciplinary Life Science research. These examples concern recently introduced drugs for the treatment of infections and neurological diseases; application of -OMICS techniques and genetic tools in medical mycology and the regulation of mycotoxin production; as well as some highlights of mushroom cultivaton in Asia. Examples for new diagnostic tools in medical mycology and the exploitation of new candidates for therapeutic drugs, are also given. In addition, two entries illustrating the latest developments in the use of fungi for biodegradation and fungal biomaterial production are provided. Some other areas where there have been and/or will be significant developments are also included. It is our hope that this paper will help realise the importance of fungi as a potential industrial resource and see the next two decades bring forward many new fungal and fungus-derived products.

CARD9 deficiency predisposing chromoblastomycosis: A case report and comparative transcriptome study.

CARD9 mutations are known to predispose patients to phaeohyphomycosis caused by different dematiaceous fungal species. In this study, we report for the first time a patient of chromoblastomycosis caused by Phialophora expanda, who harbored CARD9 mutation. Through a series of in vivo and in vitro studies, especially a comparative transcriptome study, we compared this case with our former patient suffering from phaeohyphomycosis caused by Phialophora americana. We showed that P. expanda is prone to forming sclerotic bodies both in vitro and in Card9 knockout mice, and has a stronger immunogenicity than P. americana. These data preliminary demonstrated that besides host defense, fungal specificity also contributed to the clinical phenotype in CARD9 deficient patients with dematiaceous fungal infections.

Post-translational changes in Phialophora verrucosa via lysine lactylation during prolonged presence in a patient with a CARD9-related immune disorder.

CARD9-related inherited immune disorders are a major risk factor for chronic disseminated fungal infection. In addition to pathogens of Candida and dermatophytes, the environmental opportunists of the black yeast-like fungi are relatively frequent in this patient cohort. Particularly the genus Phialophora is overrepresented. We investigated two isolates of a strain of P. verrucosa residing in a CARD9 patient, sampled with a period of ten years apart. Genomes, melanization and antifungal susceptibility of progenitor and derived strains were compared, and potential adaptation to the host habitat was investigated with proteomic techniques using post-translational modification as a proxy. Global lactylation analysis was performed using high accuracy nano-LC-MS/MS in combination with enrichment of lactylated peptides from digested cell lysates, and subsequent peptide identification. The genome of the derived isolate had accumulated 6945 SNPs, of which 31 were detected in CDS. A large number of identified proteins were significantly enriched, e.g. in melanin biosynthesis. A total of 636 lactylation sites on 420 lactylated proteins were identified, which contained in 26 types of modification motifs. Lysine lactylation (Kla) was found in 23 constituent proteins of the ribosome, indicating an impact of Kla in protein synthesis. Twelve lactylated proteins participated in pathogenicity. A protein-protein interaction (PPI) network analysis suggested that protein lactylations are widely distributed influencing various biological processes. Our findings reveal widespread roles for lysine lactylation in regulating metabolism and melanin biosynthesis in black fungi. Several large rearrangements and inversions were observed in the genome, but genomic changes could not be linked to adaptation or to known clinically relevant properties of progenitor to derived isolate; in vitro antifungal susceptibility had largely remained unaltered.

A 44-Year-Old Man With Right Limb Convulsion and Cavitary Lung Lesion With Diffuse Interstitial Abnormalities.

CASE PRESENTATION: A 44-year-old man with a history of asthma presented with intermittent convulsion of the right limb, fever in the late afternoon, and decreased exercise tolerance over 2 months. Occasional productive cough, no hemoptysis, and weight loss of nearly 6 kg were observed during this period. Neither chemotherapy nor oral immunosuppressive drugs had been administered, and no exposure to toxic substances was known. He was a cook and had smoked approximately one pack of cigarettes per day for the past 20 years. The living environment was relatively humid. The patient presented to a local hospital, where the workup was notable for low-density shadows in the left parieto-occipital lobe and a cavity in the right upper lobe of the lung with bilateral diffuse interlobular septal thickening and multiple patchy ground-glass opacities. The brain and lung lesions were 18F-fluorodeoxyglucose avid on PET/CT scan. Bronchoscopy with BAL and transbronchial biopsy were nondiagnostic. While preparing for another diagnostic procedure, the patient gradually developed increasing dyspnea and more frequent convulsions with the progression of lesions on the follow-up chest CT scan. The patient was transferred to our hospital.

Mycobacterium smegmatis Skin Infection Following Cosmetic Procedures: Report of Two Cases.

Mycobacterium smegmatis is an acid-fast bacillus of rapidly growing mycobacteria (RGM) of nontuberculous mycobacteria (NTM). M. smegmatis was considered nonpathogenic to humans until 1986, when the first patient was linked to the infection. To date, fewer than 100 cases have been reported in the literature, mainly related to various surgical procedures. Herein, we report two immunocompetent patients who acquired M. smegmatis infection following cosmetic procedures. Due to the rarity of M. smegmatis infection in routine clinical practice, it is challenging for medical providers to diagnose and treat patients with M. smegmatis infection. M. smegmatis infection should be considered for patients with chronic skin and soft tissue infections at the injection site or surgical site following cosmetic procedures. Histological findings, pathogen identification by molecular testing or bacterial culture are required to make a definitive diagnosis. Medical providers should raise awareness of M. smegmatis infection for patients with chronic skin and soft tissue infections after cosmetic procedures. Stringent sterile procedures for surgical instruments, supplies, and environments should be enforced.

Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Scedosporium, Acremonium-Like, Scopulariopsis, and Microascus Species.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a powerful microorganism identification tool. Research on MALDI-TOF MS identification of rare filamentous fungi is still lacking. This study aimed to evaluate the performance of MALDI-TOF MS in the identification of Scedosporium, Acremonium-like, Scopulariopsis, and Microascus species. Sabouraud broth cultivation and formic acid/acetonitrile protein extraction were used for MALDI-TOF MS identification by a Bruker Biotyper system. An in-house database containing 29 isolates of Scedosporium, Acremonium-like, Scopulariopsis, and Microascus spp. was constructed. A total of 52 clinical isolates were identified using the Bruker Filamentous Fungi Library v1.0 (FFL v1.0) alone, and Filamentous Fungi Library v1.0 plus the in-house library, respectively. The mass spectrum profile (MSP) dendrograms of the 28 Scedosporium isolates, 26 Acremonium-like isolates, and 27 Scopulariopsis and Microascus isolates were constructed by MALDI Biotyper OC 4.0 software, respectively. The correct species identification rate significantly improved when using the combined databases compared with that when using FFL v1.0 alone (Scedosporium spp., 75% versus 0%; Acremonium-like spp., 100% versus 0%; Scopulariopsis and Microascus spp., 100% versus 62.5%). The MSP dendrograms differentiated Acremonium-like species, Scopulariopsis and Microascus species clearly, but cannot distinguish species in the Scedosporium apiospermum complex. In conclusion, with an expanded database, MALDI-TOF MS is an effective tool for the identification of Scedosporium, Acremonium-like, Scopulariopsis, and Microascus species.

Dysbiosis of nail microbiome in patients with psoriasis.

Shifts in skin microbiome are considered to be involved in the pathogenesis of psoriasis. However, data on the microbial dysbiosis of nail psoriasis are scarce. In this study, we aim to investigate and characterize the nail bacterial and fungal microbiome in patients with psoriasis. Nail samples were collected prospectively from 36 subjects with nail psoriasis, 24 psoriatic subjects without nail involvement and 32 healthy controls. Amplicon sequencing was performed to evaluate the bacterial and fungal community compositions. Significant alterations in the bacterial microbiome were found in the nail samples of psoriatic patients. The unaffected nails in psoriatic patients were associated with higher bacterial diversity, and a higher relative abundance of Enhydrobacter, whereas nail psoriasis was correlated with a decreased relative abundance of Anaerococcus. Shifts in fungal community composition were reflected by a higher proportion of Malassezia in the unaffected nails of psoriatic patients and an increased proportion of Candida in psoriatic nails. Shifts in the nail microbiome in psoriasis suggest a potential role of microbes in the development of nail psoriasis. Future researches focusing on these microorganisms may help to explain the pathogenesis of psoriasis.

Ketoconazole 2% cream alters the skin fungal microbiome in seborrhoeic dermatitis: a cohort study.

BACKGROUND: Seborrhoeic dermatitis (SD) is a common chronic inflammatory dermatosis. Current theories on the pathogenesis of SD highlight the role of microbes on the skin surface. Ketoconazole is commonly used for the treatment of SD; however, there are limited data focusing on the effects of ketoconazole in shaping the skin microbiome in patients with SD. AIM: In this prospective cohort study, we used a high-throughput DNA sequencing method to characterize the cutaneous microbial communities of patients with SD before and after topical ketoconazole treatment. METHODS: In total, 30 patients with facial SD and 15 age- and sex-matched healthy controls (HCs) were enrolled in this study. Skin swabs were collected from SD lesional sites of the cheek at baseline, after ketoconazole treatment and 2 weeks post-treatment. DNA was extracted from skin samples. The bacterial 16S V3V4 rRNA and fungal internal transcribed spacer 1-5F regions were sequenced, and the microbial community compositions were analysed. RESULTS: Significantly lower bacterial and fungal diversities were detected at the lesional sites of facial SD compared with HCs. A decreased relative abundance of Cutibacterium and increased abundances of Malassezia and Staphylococcus were found in facial SD. Disease diversity was positively correlated with the relative abundances of Malassezia, Staphylococcus and Corynebacterium, while transepidermal water loss was negatively associated with the relative abundance of Cutibacterium. After ketoconazole treatment, fungal Shannon diversity and the relative abundances of Candida and Aspergillus were significantly increased at the lesional sites, and the relative abundance of Malassezia showed a decreasing trend. These changing trends were maintained until 2 weeks post-treatment. CONCLUSION: Facial SD showed lower fungal diversity accompanied by increased relative abundances of Malassezia and Staphylococcus and decreased relative abundance of Cutibacterium. Ketoconazole treatment reduced Malassezia and increased fungal diversity to restore skin microbial communities.