The phenotype and prediction of long-term physical, mental and cognitive COVID-19 sequelae 20 months after recovery, a community-based cohort study in China.

Long-term sequelae clustering phenotypes are important for precise health care management in COVID-19 survivors. We reported findings for 1000 survivors 20 months after diagnosis of COVID-19 in a community-based cohort in China. Sequelae symptoms were collected from a validated questionnaire covering 27 symptoms involved in five organ systems including self-reported physical condition, dyspnea, cognitive function and mental health. The generalized symptoms were reported with the highest rate (60.7%), followed by the mental (48.3%), cardiopulmonary (39.8%), neurological (37.1%; cognitive impairment, 15.6%), and digestive symptoms (19.1%). Four clusters were identified by latent class analysis: 44.9% no or mild group (cluster 1), 29.2% moderate group with mainly physical impairment (cluster 2), 9.6% moderate group with mainly cognitive and mental health impairment (cluster 3), and 16.3% severe group (cluster 4). Physical comorbidities or history of mental disorders, longer hospitalization periods and severe acute illness predicted severe group. For moderate group, adults less than 60 years, with physical comorbidities and severe acute illness were more likely to have physical symptoms, while adult women with longer hospitalization stays had increased risk of cognitive and mental health impairment. Overall, among more than half of community COVID-19 survivors who presented moderate or severe sequelae 20 months after recovery, three-tenth had physical vulnerability that may require physical therapy aiming to improve functioning, one-tenth mental or cognitive vulnerable cases need psychotherapy and cognitive rehabilitation, and one-sixth severe group needs multidisciplinary clinical management. The remaining half is free to clinical intervention. Our findings introduced an important framework to map numerous symptoms to precise classification of the clinical sequelae phenotype and provide information to guide future stratified recovery interventions.

A systematic review and meta-analysis of long term physical and mental sequelae of COVID-19 pandemic: call for research priority and action.

The long-term physical and mental sequelae of COVID-19 are a growing public health concern, yet there is considerable uncertainty about their prevalence, persistence and predictors. We conducted a comprehensive, up-to-date meta-analysis of survivors' health consequences and sequelae for COVID-19. PubMed, Embase and the Cochrane Library were searched through Sep 30th, 2021. Observational studies that reported the prevalence of sequelae of COVID-19 were included. Two reviewers independently undertook the data extraction and quality assessment. Of the 36,625 records identified, a total of 151 studies were included involving 1,285,407 participants from thirty-two countries. At least one sequelae symptom occurred in 50.1% (95% CI 45.4-54.8) of COVID-19 survivors for up to 12 months after infection. The most common investigation findings included abnormalities on lung CT (56.9%, 95% CI 46.2-67.3) and abnormal pulmonary function tests (45.6%, 95% CI 36.3-55.0), followed by generalized symptoms, such as fatigue (28.7%, 95% CI 21.0-37.0), psychiatric symptoms (19.7%, 95% CI 16.1-23.6) mainly depression (18.3%, 95% CI 13.3-23.8) and PTSD (17.9%, 95% CI 11.6-25.3), and neurological symptoms (18.7%, 95% CI 16.2-21.4), such as cognitive deficits (19.7%, 95% CI 8.8-33.4) and memory impairment (17.5%, 95% CI 8.1-29.6). Subgroup analysis showed that participants with a higher risk of long-term sequelae were older, mostly male, living in a high-income country, with more severe status at acute infection. Individuals with severe infection suffered more from PTSD, sleep disturbance, cognitive deficits, concentration impairment, and gustatory dysfunction. Survivors with mild infection had high burden of anxiety and memory impairment after recovery. Our findings suggest that after recovery from acute COVID-19, half of survivors still have a high burden of either physical or mental sequelae up to at least 12 months. It is important to provide urgent and appropriate prevention and intervention management to preclude persistent or emerging long-term sequelae and to promote the physical and psychiatric wellbeing of COVID-19 survivors.

Proteome-Wide Analysis of Lysine 2-Hydroxyisobutyrylation in Aspergillus fumigatus.

Aspergillus fumigatus is the significant causative agent in cases of invasive aspergillosis, leading to a high mortality rate in immunocompromised patients. A comprehensive understanding of its growth patterns and metabolic processes within the host is a critical prerequisite for the development of effective antifungal strategies. Lysine 2-hydroxyisobutyrylation (Khib) is a highly conserved protein posttranslational modifications (PTM) found in various organisms. In this study, we investigate the biological impact of Khib in A. fumigatus. Using a combination of antibody enrichment with the conventional LC-MS/MS method, the pattern of Khib-modification in proteins and their respective sites were analyzed in a wild type strain of A. fumigatus. Our findings revealed 3494 Khib-modified proteins with a total of 18,091 modified sites in this strain. Functional enrichment analysis indicated that these Khib-modified proteins participate in a diverse range of cellular functions, spanning various subcellular locations such as ribosome biosynthesis, protein synthesis and nucleocytoplasmic transport. Notably, when compared with other reported eukaryotes, A. fumigatus exhibited consistently higher numbers of Khib-modified proteins, suggesting the potential significance of this modification in this organism. An interesting observation is the prevalence of Khib modifications in most enzymes involved in the ergosterol synthesis pathway. The insights gathered from this study provide new avenue for studying PTM-associated mechanisms in fungal growth and offer potential implication for antifungal drug development.

A novel model combining genes associated with disulfidptosis and glycolysis to predict breast cancer prognosis, molecular subtypes, and treatment response.

Breast cancer (BC) is a heterogeneous malignancy with a dismal prognosis. Disulfidptosis is a novel type of regulated cell death that happens in the presence of glucose deficiency and is linked to the metabolic process of glycolysis. However, the mechanism of action of disulfidptosis and glycolysis-related genes (DGRG) in BC, as well as their prognostic value in BC patients, remain unknown. After identifying the differentially expressed DGRG in normal and BC tissues, a number of machine learning algorithms were utilized to select essential prognostic genes to develop a model, including SLC7A11, CACNA1H, SDC1, CHST1, and TFF3. The expression characteristics of these genes were then examined using single-cell RNA sequencing, and BC was classified into three clusters using "ConsensusClusterPlus" based on these genes. The DGRG model's median risk score can categorize BC patients into high-risk and low-risk groups. Furthermore, we investigated variations in clinical landscape, immunoinvasion analysis, tumor immune dysfunction and rejection (TIDE), and medication sensitivity in patients in the DGRG model's high- and low-risk groups. Patients in the low-risk group performed better on immunological and chemotherapeutic therapies and had lower TIDE scores. In conclusion, the DGRG model we developed has significant clinical application potential because it can accurately predict the prognosis of BC, TME, and pharmacological treatment responses.

Comparative genomics predict specific genes in potential mucorales identification.

Mucoralean fungi could cause mucormycosis in humans, particularly in immunodeficient individuals and those with diabetes mellitus or trauma. With plenty of species and genera, their molecular identification and pathogenicity have a large deviation. Reported cases of mucormycosis showed frequent occurrence in Rhizopus species, Mucor species, and Lichtheimia species. We analyzed the whole genome sequences of 25 species of the top 10 Mucorales genera, along with another 22 important pathogenic non-Mucorales species, to dig the target genes for monitoring Mucorales species and identify potential genomic imprints of virulence in them. Mucorales-specific genes have been found in various orthogroups extracted by Python script, while genus-specific genes were annotated covering cellular structure, biochemistry metabolism, molecular processing, and signal transduction. Proteins related to the virulence of Mucorales species varied with distinct significance in copy numbers, in which Orthofinder was conducted. Based on our fresh retrospective analysis of mucormycosis, a comparative genomic analysis of pathogenic Mucorales was conducted in more frequent pathogens. Specific orthologs between Mucorales and non-Mucoralean pathogenic fungi were discussed in detail. Referring to the previously reported virulence proteins, we included more frequent pathogenic Mucorales and compared them in Mucorales species and non-Mucorales species. Besides, more samples are needed to further verify the potential target genes.

Efficacy and secondary infection risk of tocilizumab, sarilumab and anakinra in COVID-19 patients: A systematic review and meta-analysis.

As the pandemic progresses, the pathophysiology of coronavirus disease 2019 (COVID-19) is becoming clearer and the potential for immunotherapy is increasing. However, clinical efficacy and safety of immunosuppressants (including tocilizumab, sarilumab and anakinra) treatment in COVID-19 patients are not yet known. We searched PubMed, Embase Medline, Web of Science and MedRxiv using specific search terms in studies published from 1 January 2020 to 20 December 2020. In total, 33 studies, including 3073 cases and 6502 controls, were selected for meta-analysis. We found that immunosuppressant therapy significantly decreased mortality in COVID-19 patients on overall analysis (odds ratio = 0.71, 95% confidence interval = 0.57-0.89, p = 0.004). We also found that tocilizumab and anakinra significantly decreased mortality in patients without any increased risk of secondary infection. In addition, we found similar results in several subgroups. However, we found that tocilizumab therapy significantly increased the risk of fungal co-infections in COVID-19 patients. This represents the only systematic review and meta-analysis to investigate the efficacy and secondary infection risk of immunosuppressant treatment in COVID-19 patients. Overall, immunosuppressants significantly decreased mortality but had no effect on increased risk of secondary infections. Our analysis of tocilizumab therapy showed a significantly increased risk of fungal co-infections in these patients.

The Domestic Isolation of Terbinafine- and Itraconazole-Resistant Trichophyton indotineae in Chinese Mainland.

BACKGROUND: Trichophyton indotineae, a new species of dermatophytes, has become a significant concern in treating dermatophytosis due to the high level of terbinafine resistance reported in India and even worldwide. OBJECTIVES: This study aimed to report the terbinafine- and itraconazole-resistant T. indotineae in Chinese mainland, by identifying the phylogenetic classification of the isolate strain, and detecting the drug resistance, gene mutation and expression. PATIENTS/METHODS: The skin scales of the patient were cultured on SDA and the isolate was authenticated by DNA sequencing and MALDI-TOF MS. Antifungal susceptibility testing was performed following the M38-A2 CLSI protocol to examine the MICs values of terbinafine, itraconazole, fluconazole, etc. The strain was screened for mutations in the squalene epoxidase (SQLE) gene by Sanger sequencing and detected the expression of CYP51A and CYP51B by qRT-PCR. RESULTS: A multi-resistant ITS genotype VIII sibling of the T. mentagrophytes complex (T. indotineae) was isolated in Chinese mainland. The strain harbored high terbinafine MIC of > 32 µg/mL and itraconazole MIC of 1.0 µg/mL, which was identified a mutation in the squalene epoxidase gene with amino acid substitution (Phe397Leu, mutation 1191C > A). In addition, overexpression of CYP51A and CYP51B was observed. With multiple relapses, the patient finally achieved clinical cure by itraconazole pulse therapy and topical clotrimazole cream for 5 weeks. CONCLUSIONS: The first domestic strain of terbinafine- and itraconazole-resistant T. indotineae from a patient in Chinese mainland was isolated. Itraconazole pulse therapy can be an effective method for the treatment of T. indotineae.

In Vitro Antifungal Activity of Azoles and Other Antifungal Agents Against Pathogenic Yeasts from Vulvovaginal Candidiasis in China.

BACKGROUND: Vulvovaginal candidiasis (VVC) is a public health issue worldwide. Little is known of the optimal treatment of recurrent VVC (RVVC) has not been established. OBJECTIVE: Through the in vitro antifungal susceptibility profiling of VVC isolates, we hope to foster significant improvements in the control and treatment of this disease. METHODS: Candida isolates from VVC patients were collected from 12 hospitals in 10 cities across China. Species were identified by phenotype analysis and DNA sequencing. Species were identified by phenotype analysis and DNA sequencing. Susceptibilities to 11 drugs were determined by Clinical and Laboratory Standards Institute broth microdilution. RESULTS: 543 strains were isolated from those VVC patients enrolled in this study, of which, 15.7% were from RVVC. The most commonly identified species was C. albicans (460, 84.71%), and the most commonly non-albicans Candida spp. (NAC) was C. glabrata (47, 8.66%). NAC also included C. Krusei, Meyerozyma Guillermondii, Meyerozyma Caribbica, C. Tropicalis, C. Parapsilosis, and C. Nivariensis. Most C. albicans isolates were susceptible to caspofungin (99.8%), followed by fluconazole (92%) and voriconazole (82.6%). The proportion of C. albicans strains with wild type (WT) MICs that were susceptible to amphotericin B and caspofungin were 98%, followed by posaconazole at 95%, itraconazole at 86%, fluconazole at 74% and voriconazole at 54%. The fluconazole MICs for C. albicans were lower than those for NAC (P < 0.05), while the itraconazole MICs showing no significant difference (P > 0.05). The susceptible rate of uncomplicated VVC to fluconazole was 92%. The proportion of WT strains to fluconazole in RVVC was much lower than that in other types of VVC (67 vs. 77%, P < 0.05). However, the proportions of WT strains to itraconazole in RVVC was over 85%, which was much higher than that to fluconazole (87 vs. 67%, P < 0.05). CONCLUSIONS: C. albicans was still the predominant pathogen for VVC in China, while C. glabrata was the main species in NAC. Fluconazole could still be used as an empirical treatment for uncomplicated VVC. However, fluconazole may not be the first choice for the therapy of RVVC. In such cases, itraconazole appears to be the more appropriate treatment. As for VVC caused by NAC, nonfluconazole drugs, such as itraconazole, may be a good choice.

Retrieving a disrupted gene encoding phospholipase A for fibre enhancement in allotetraploid cultivated cotton.

After polyploidization originated from one interspecific hybridization event in Gossypium, Gossypium barbadense evolved to produce extra-long staple fibres than Gossypium hirsutum (Upland cotton), which produces a higher fibre yield. The genomic diversity between G. barbadense and G. hirsutum thus provides a genetic basis for fibre trait variation. Recently, rapid accumulation of gene disruption or deleterious mutation was reported in allotetraploid cotton genomes, with unknown impacts on fibre traits. Here, we identified gene disruptions in allotetraploid G. hirsutum (18.14%) and G. barbadense (17.38%) through comparison with their presumed diploid progenitors. Relative to conserved genes, these disrupted genes exhibited faster evolution rate, lower expression level and altered gene co-expression networks. Within a module regulating fibre elongation, a hub gene experienced gene disruption in G. hirsutum after polyploidization, with a 2-bp deletion in the coding region of GhNPLA1D introducing early termination of translation. This deletion was observed in all of the 34 G. hirsutum landraces and 36 G. hirsutum cultivars, but not in 96% of 57 G. barbadense accessions. Retrieving the disrupted gene GhNPLA1D using its homoeolog GhNPLA1A achieved longer fibre length in G. hirsutum. Further enzyme activity and lipids analysis confirmed that GhNPLA1A encodes a typical phospholipase A and promotes cotton fibre elongation via elevating intracellular levels of linolenic acid and 34:3 phosphatidylinositol. Our work opens a strategy for identifying disrupted genes and retrieving their functions in ways that can provide valuable resources for accelerating fibre trait enhancement in cotton breeding.

Diagnosis accuracy of Raman spectroscopy in the diagnosis of breast cancer: a meta-analysis.

To investigate the diagnostic efficiency of Raman spectroscopy for the diagnosis of breast cancer, we searched PubMed, Web of Science, Cochrane Library, and Embase for articles published from the database establishment to May 20, 2022. Pooled sensitivity, specificity, diagnostic odds ratio, and area under the receiver pooled operating characteristic curve were derived for the included studies as outcome measures. The methodological quality was assessed according to the questionnaires and criteria suggested by the Diagnostic Accuracy Research Quality Assessment-2 tool. Sixteen studies were included in this meta-analysis. The pooled sensitivity and specificity of Raman spectroscopy for breast cancer diagnosis were 0.97 (95% CI, [0.92-0.99]) and 0.96 (95% CI, [0.91-0.98]). The diagnostic odds ratio was 720.89 (95% CI, [135.73-3828.88]) and the area under the curve of summary receiver operating characteristic curves was 0.99 (95% CI, [0.98-1]). Subgroup analysis revealed that all subgroup types in our analysis, including different races, sample types, diagnostic algorithms, number of spectra, instrument types, and laser wavelengths, turned out to have a sensitivity and specificity greater than 0.9. Significant heterogeneity was found between studies. Deeks' funnel plot demonstrated that publication bias was acceptable. This meta-analysis suggests that Raman spectroscopy may be an effective and accurate tool to differentiate breast cancer from normal breast tissue, which will help us diagnose and treat breast cancer.

Characterization of mitogenomes from four Mucorales species and insights into pathogenicity.

BACKGROUND: Mucorales, as one major order of Zygomycetes fungi, can infect human beings and cause serious consequence. We have noticed the pathogenicity of Mucorales is closely related to energy metabolism, while mitochondria play the role of energy factories in almost all biological activities. METHODS: Virulence of M irregularis, M hiemalis, L corymbifera and R arrhizus were verified in Galleria mellonella larvae, as well as mitochondrial gene copies analysed with RT-qPCR. Mitogenomes of the four Mucorales species were sequenced based on illumina NovaSeq technology to study their characteristic features and functional regions. RESULTS: Variant virulence of M irregularis, M hiemalis, L corymbifera and R arrhizu were verified by clinical retrospective data and our G mellonella infection models, also copies of mitochondrial genes indicated the significant associations with pathogenicity. A total of 274.18 clean reads were generated to be assembled; the complete mitogenomes of the four Mucorales species were obtained with totally different length. After the genomes annotated and compared, M irregularis was found more similar with M hiemalis than those of L corymbifera and R arrhizus, especially the small (rrns) and large (rrnl) subunits of mitochondrial ribosomal RNA (rRNA) genes. The GC content, ncRNAs and the distribution of the SNPs and InDels were also compared, and the GC content rate of fungi seems to be related to the fungal thermal adaptability. In addition, linear mitogenomes of these four Mucorales showed diverse arrangements of orf genes and directionality of some conserved gene elements. CONCLUSION: This study uncovered the pathogenicity variances among the four Mucorales species and the relationship between their mitogenomic features and clinical pathogenicity. Further studies like spatial structure of mitochondrial genomes and the comprehensive analysis of transcription regulation are needed.

A thermal adaptation landscape related to virulence in Mucor irregularis transcriptional profiles.

OBJECTIVES: Our study aimed to better understand the different thermal adaptation in Mucor irregularis (M. irregularis) strains under high temperature and the involved virulence-related genes, and to offer more appropriate explanation for the diverse pathogenicity of M. irregularis in human infections. METHODS: M. irregularis isolates were incubated at 30 and 35°C for Illumina HiSeq technology (RNA-seq), as well as the virulence difference detected through Galleria mellonella infection models. We verified their transcriptional profile with RT-PCR and analysed differentially expressed genes with GO and KEGG annotations. RESULTS: All 25 isolates formed the biggest colonies at 28°C and did not grow at 37°C, while were differently inhibited at 22 and 35°C. Six selected M. irregularis displayed virulence in sync with their growth condition at high temperature. From the outcomes of RNA-seq, a total of 1559 differentially expressed genes (FC ≥ 2, FDR < 0.05) were obtained, of which 1021 genes were upregulated, and 538 genes were downregulated. Cell wall structure genes related to Ras-like and GH16 proteins, influx-efflux pumps consist of transmembrane proteins as ABC and MFS proteins, and metabolic genes as DGKɛ and Hsfs, seem to be essential in thermal adaptation and virulence of M. irregularis. CONCLUSION: We found some common genes expressed at high temperature, while some others specifically related to M. irregularis isolates with different virulence and thermal adaptation. Further research of genes involved in the pathogenic process is needed for the development of potential targeted antifungal.

Divergent improvement of two cultivated allotetraploid cotton species.

Interspecific genomic variation can provide a genetic basis for local adaptation and domestication. A series of studies have presented its role of interspecific haplotypes and introgressions in adaptive traits, but few studies have addressed their role in improving agronomic character. Two allotetraploid Gossypium species, Gossypium barbadense (Gb) and G. hirsutum (Gh) originating from the Americas, are cultivated independently. Here, through sequencing and the comparison of one GWAS panel in 229 Gb accessions and two GWAS panels in 491 Gh accessions, we found that most associated loci or functional haplotypes for agronomic traits were highly divergent, representing the strong divergent improvement between Gb and Gh. Using a comprehensive interspecific haplotype map, we revealed that six interspecific introgressions from Gh to Gb were significantly associated with the phenotypic performance of Gb, which could explain 5%-40% of phenotypic variation in yield and fibre qualities. In addition, three introgressions overlapped with six associated loci in Gb, indicating that these introgression regions were under further selection and stabilized during improvement. A single interspecific introgression often possessed yield-increasing potential but decreased fibre qualities, or the opposite, making it difficult to simultaneously improve yield and fibre qualities. Our study not only has proved the importance of interspecific functional haplotypes or introgressions in the divergent improvement of Gb and Gh, but also supports their potential value in further human-mediated hybridization or precision breeding.

Comprehensive genome and transcriptome analysis of the dermatophyte Trichophyton schoenleinii reveals the candidate pathogenic genes.

BACKGROUND: Trichophyton schoenleinii is an anthropophilic dermatophyte that causes tinea favosa. Nowadays, it remains an important pathogen in some regions of the world, mainly epidemic in Africa and West Asia. Despite the medical importance of T. schoenleinii infections, a high-quality reference genome for T. schoenleinii is still unavailable, neither its transcriptomic profile. OBJECTIVES: The aim of the current study was to improve understanding of the underlying pathogenic mechanism of T. schoenleinii, and to define the candidate pathogenic genes of T. schoenleinii. METHODS: Comprehensive genomic analysis of T. schoenleinii was carried out by Illumina and PacBio sequencing platforms. Transcriptome profiles of T. schoenleinii cultured in vitro in two media containing either keratin or soy protein were determined using RNA sequencing (RNA-seq) technology. RESULTS: Here, we present the first draft genome sequence of T. schoenleinii strain T2s, which consists of 11 scaffolds containing 7474 predicted genes. Transcriptome analysis showed that genes involved in keratin hydrolysis have higher expression in T. schoenleinii grown in keratin medium, including genes encoding proteases, cysteine dioxygenase and acetamidase. Other genes with higher expression include genes encoding the components of the pH-responsive signal transduction pathways and transcription factors, many of which may play a role in pathogenicity. CONCLUSION: In summary, this study provides new insights into the pathogenic mechanism of T. schoenleinii and highlights candidate genes for further development of novel targets in disease diagnosis and treatment of tinea favosa.

Comparative Genomics and Molecular Analysis of Epidermophyton floccosum.

Epidermophyton floccosum is one of the most common agents of human superficial fungal infections, compared with genus Trichophyton and Microsporum, it possesses uniqueness in ecology traits and rarely causing hair infections. E. floccosum is so far the only representative species of genera Epidermophyton, and it is known as anthropophilic dermatophytes. To further reveal the genome sequences and clues of virulence factors, thus in this study, we sequenced the genome of E. floccosum (CGMCC (F) E1d), and performed comparative genomic analysis with other dermatophytes. It is revealed that E. floccosum owns the largest genome size and similar GC content compared with other dermatophytes. A total of 7565 genes are predicted. By comparing with the closest species N. gypseum, our study reveals that number and structure of adhesion factors, secreted proteases and LysM domain might contribute to the pathogenic and ecological traits of E. floccosum. Mating genes is also detected in genome data. Furthermore, we performed AFLP analysis trying to discuss intraspecific differences of E. floccosum, but no significant relationship is found between genotype and geographical distribution. Upon above, our study provides a deeper understanding and strong foundation for future researches about E. floccosum.

In Vitro Activities of Ravuconazole and Isavuconazole against Dematiaceous Fungi.

The in vitro activities of 11 antifungals against 84 dematiaceous fungi were tested. For most tested fungal species, the MIC values of ravuconazole and isavuconazole were lower than those obtained with itraconazole, voriconazole, and posaconazole. Ravuconazole and isavuconazole appear to be more efficient against most dematiaceous fungal infections than the other triazoles. However, some pigmented fungi, such as Bipolaris spicifera and Veronaea botryosa, remain more susceptible to other triazoles or to echinocandins.

Auxotrophic mutations of Trichophyton rubrum created by in vitro synthesized Cas9 ribonucleoprotein.

BACKGROUND: Trichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80-90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum. RESULTS: The gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method. We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing. CONCLUSIONS: We developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species.

Assembly and analysis of the whole genome of Arthroderma uncinatum strain T10, compared with Microsporum canis and Trichophyton rubrum.

BACKGROUND: Arthroderma uncinatum is a geophilic dermatophyte that occasionally causes superficial infections in humans leading to skin diseases. OBJECTIVES: To better understand the ecology and potential pathogenicity of A uncinatum, we analysed its whole genome. We compared A uncinatum with the genome of the zoophilic dermatophyte Microsporum canis and with the anthropophilic species Trichophyton rubrum. The compared species differ significantly in the frequency of human infection. METHODS: We reported the genome sequence of strain T10 of A uncinatum based on SMRT (single-molecule real-time) technology (PacBio). RESULTS: We obtained a near-complete 23.56 Mb genome, with 7153 predicted gene models and ~20% repetitive sequences. We subsequently determined the specific genetic differences between A uncinatum, M canis and T rubrum. The functional enrichment analysis suggests that A uncinatum is particularly enriched in specific virulence genes. This suggests that the ancestral condition in dermatophytes is with high virulence, which has decreased in the course of evolution to enhance coexistence with animal or human hosts.

Molecular Identification and In Vitro Antifungal Susceptibility of Aspergillus Isolates Recovered from Otomycosis Patients in Western China.

Aspergillus species are the most common causative agents involved in otomycosis. In this study, 45 Aspergillus isolates were obtained from patients with otomycosis in western China during 2013-2016. The aim of this study is to identify the Aspergillus isolates to the species level by using ß-tubulin gene sequencing and to evaluate their in vitro susceptibility to nine antifungal drugs: amphotericin B, itraconazole, voriconazole, posaconazole, ravuconazole, isavuconazole, caspofungin, micafungin and anidulafungin according to CLSI M38-A2. Our results indicate that A. tubingensis (18/45) is the predominant Aspergillus species causing ear infections in western China, which is three times more than its sibling species A. niger (6/45) and A. welwitschiae (2/45). Other detected species were A. fumigatus (n = 8), A. terreus (n = 7) and A. flavus (n = 4). Antifungal susceptibility data indicate that triazoles and echinocandins are active against the most Aspergillus isolates. There are no significant differences in the susceptibility among A. niger, A. tubingensis and A. welwitschiae to each of the antifungals tested. One azole-resistant A. fumigatus isolate with a TR34/L98H mutation in the CYP51A gene and one posaconazole-resistant A. terreus isolate presented among the studied isolates. In conclusion, A. tubingensis is the most prevalent Aspergillus species causing otomycosis in western China. Posaconazole and echinocandins are potential drugs for treatment of otomycosis due to Aspergillus; however, in vivo efficacy remains to be determined.

Whole-genome resequencing of Trichophyton rubrum provides insights into population differentiation and drug resistance.

Trichophyton rubrum (T. rubrum) is anthropophilic fungus and thus a very common cause of dermatophyte infections around the world. Infection of T. rubrum could result in conditions such as tinea capitis, tinea corporis, tinea inguinalis, tinea manus, tinea unguium, or tinea pedis. Because of this, the resistance of T. rubrum to antifungal therapies has drawn extensive research interest. However, the pathogenic characteristics of T. rubrum, such as site of infections, geographic location and host groups, have yet to be explored. In this study, the whole genome of 48 strains from different regions is resequenced and the population structure and association of single nucleotide polymorphism with resistance to six widely used antifungal drugs are analyzed. A total of 23,394 genomic variations are detected, which cover 2165 genes with only 15.14% of the variations located in exons. The population structure of T. rubrum is monomorphic, and genetic diversity is very low. Population structure analysis shows that the 48 sampled strains can be divided into two sub-populations. The gene TERG_08771 harboring the highest SNPs density is found to be associated with resistance to voriconazole. Although many proteins have yet to be identified and explored, association studies could still be useful to identify drug resistance or drug-susceptible loci, which would warrant further insightful investigations.