Mycopathologia最新文献

Numbers of infections with azole-resistant Aspergillus fumigatus (ARAf) were rising in the last decades. We assessed ARAf susceptibility trends towards five antifungal agents (amphotericin B (AMB), itraconazole (ITR), voriconazole (VCZ), olorofim (OLO) and manogepix (MGX)) over twelve years in a German Excellence Center for Medical Mycology (ECMM). In addition, underlying mutations were studied and correlated with trends in minimum inhibitory concentration (MIC). Broth microdilution (BMD) was performed following EUCAST guidelines for 143 clinical ARAf isolates collected between the years 2011 and 2022 in a West German tertiary care centre. BMD was carried out for all antifungal agents in the following concentration ranges: 0.016-8 mg/L for AMB, ITR and VCZ as well as 0.001-0.5 mg/L for OLO and 0.004-2 mg/L for MGX. Molecular assays on mutations associated with antifungal resistance were performed for all 143 isolates (AsperGenius® 1.0, Pathonostics, Maastricht, The Netherlands) and for a total of ten non TR₃₄/L98H and TR₄₆/Y121F/T289A mutated ARAf isolates additional cyp51A sequencing was carried out. For all isolates, microdilution revealed a MIC₅₀ of > 8 mg/L for ITR, 4 mg/L for VCZ, 0.03 mg/L for OLO, 0.016 mg/L for MGX, and 0.5 mg/L for AMB. Considering EUCAST breakpoints, 97.9% of the strains (n = 140) were resistant to VCZ, 1.4% (n = 2) towards AMB and 92.3% towards ITR (n = 132). Molecular assays revealed 123 (86%) isolates with the azole resistance underlying mutation TR₃₄/L98H, 10 (7%) with a TR₄₆/Y121F/T289A mutation and 10 (7%) with other cyp51A mutations. A comparison of triazole MICs of isolates collected from 2011 to 2019 with the MICs of isolates collected between 2020 and 2022 revealed no significant differences for itraconazole (p = 0.543) and for voriconazole (p = 0.148),with a trend of increased geometric mean for ITR and VCZ MICs over time. MICs for OLO and MGX did not significantly differ between isolates with the distinct azole-resistance underlying mutations. Before 2016, the azole resistance underlying mutations were mainly TR₃₄/L98H, but the portion of isolates with TR₄₆/Y121F/T289A and other Cyp51A mutated isolates increased afterwards. We showed almost stable MICs for ITR and VCZ over twelve years in ARAf isolates from West Germany while occurring azole resistance underlying mutations varied with an increase in the proportion of TR₄₆/Y121F/T289A and other Cyp51A mutations after 2016.

Trichosporon austroamericanum is a recently described species recognized for its emerging clinical significance in invasive trichosporonosis. In this study, we present the nanopore long-read-based de novo genome assembly of the type-strain CBS 17435. Additionally, we performed genomic comparative analyses with its closest relative, Trichosporon inkin.

Trichophyton indotineae, formerly known as T. mentagrophytes internal transcribed spacer (ITS) genotype VIII, has been recognized over the last decade due to its high virulence and resistance to treatment. Its accurate identification in routine mycology laboratories remains challenging, as it shares phenotypic traits and substantial rDNA ITS similarity with T. mentagrophytes and T. interdigitale. This study aimed to identify more divergent and stable sequences via whole-genome comparisons between T. indotineae and T. interdigitale to facilitate highly specific targeting of T. indotineae using a validated quantitative polymerase chain reaction (qPCR)-based method. Our whole-genome comparison revealed at least 22 unique sequences of T. indotineae compared to T. interdigitale and revealed the divergence of the former from the reference genomes of other Trichophyton species. Among these, a 499 bp segment was identified as the most genetically distinct sequence within the T. indotineae genome. Seventy-three dermatophyte strains [T. indotineae (n = 66), non-T. indotineae (n = 7)], were tested using our qPCR assay targeting the above-mentioned stable 499-bp region. Regarding analytical performance, our T. indotineae-specific qPCR assay exhibited high sensitivity (93.3%) and specificity (100%), with a detection limit of ~ 15 genomic copies. Our approach has the potential to establish highly sensitive and specific qPCR assays without relying on specialized assay designs for single nucleotide polymorphisms in the ITS or other loci. This approach offers a practical solution for updating molecular diagnostics, particularly for novel taxa such as T. indotineae, for which limited gene data are available in public databases.

Background: Mucormycosis, a fungal emergency, poses a serious threat to both COVID-19 and non-COVID-19 individuals due to its invasive nature, rapid progression, and high rates of morbidity and mortality. This underscores the crucial need for timely detection and management. In this study, we investigated the utility of real-time PCR (RT-qPCR) assays for detecting Mucorales in clinical specimens, and assessed the performance of both SYBR Green and TaqMan probe RT-qPCR in amplifying Mucorales-specific 18S rDNA genes. We conducted accuracy analyses using direct examination with KOH as a standard for the laboratory diagnosis of mucormycosis. Additionally, we compared the results with culture and duplex PCR.

Patients/methods: Both SYBR Green and TaqMan RT-qPCR were optimized using Mucorales-specific oligonucleotides to amplify the conserved 18S rDNA targets. DNAs extracted from 120 rhino sinus specimens, which all were collected from COVID-19 patients upon suspicion of invasive fungal infections, were used for molecular diagnosis. The results of both RT-qPCR assays were compared with the result of direct microscopy, culture, and duplex Mucorales-specific PCR assay.

Results: SYBR Green real-time PCR produced a distinct melting temperature (Tm) pattern (80.24 ± 0.70 °C) and detected Mucorales in 51 out of 120 clinical samples. When compared to direct examination with KOH, the standard method for diagnosing mucormycosis, SYBR Green PCR demonstrated a sensitivity of 91.67% (95% confidence interval (CI): 86.7-96.5%) and a specificity of 90.28% (95% CI: 84.9-95.5%). In contrast, TaqMan-probe PCR identified Mucorales in 34 out of 120 samples, with a sensitivity of 64.58% (95% CI: 56-73.1%) and a specificity of 95.83% (95% CI: 92.26-99.39%).

Conclusion: SYBR Green-based PCR can be used as a reliable confirmatory test for diagnosing mucormycosis, particularly in cases with atypical hyphae, mixed infections (featuring both septate and non-septate hyphae), or when the direct examination is positive but culture results are negative. The lower sensitivity of the TaqMan-probe PCR may be attributed to factors such as using a degenerate probe, which can lead to false-negative results.

Background: Inherited genetic deficiencies in the Caspase-associated recruitment domain-containing protein 9 (CARD9) lead to increased susceptibility of patients to opportunistic melanized fungi. Such infections are recalcitrant, and the fungus possibly acquires resistance under therapy.

Objective: To evaluate differences of in vitro antifungal susceptibility of strains of melanized fungi originating from patients with CARD9 deficiency versus strains from chronic patients with unclear genetic background.

Methods: We analyzed a total of 118 isolates, including 33 from patients with CARD9 deficiency, 80 from chronic patients with other undefined immunological features, and 5 environmental strains, all collected between 1997 and 2021. All isolates were identified by sequencing the ITS spacer of the rDNA operon. Broth microdilution susceptibility tests were performed according to CLSI guidelines (M38-A3document).

Results: MIC ranges of strains from infected patients having CARD9 deficiency and other individuals were mostly similar. However, comparing these two groups, the GM MICs of posaconazole, amphotericin B and fluconazole in the CARD9 group were statistically higher and the GM MICs of terbinafine lower than those of undefined genetic background group. The FICI of the CARD9 group were higher than those of the undefined group in the combination of caspofungin plus amphotericin B and amphotericin B plus fluconazole, but lower than the undefined group in the combination of itraconazole plus terbinafine.

Conclusions: The GM MICs for posaconazole, amphotericin B, and fluconazole were significantly elevated in the CARD9 group compared to the group with undefined chronic infections. For patients with refractory infections, conducting susceptibility testing before treatment can optimize the selection of the most effective therapeutic agent, and the combination therapy of caspofungin with amphotericin B or itraconazole may be considered the preferred treatment option.

Understanding the ecological characteristics and environmental factors of migratory songbirds is essential for their conservation as well as pathogen management that may cross ecological and political boundaries. In this study, we conducted a bird trapping and banding survey and report on fungal DNA detected from birds with putative fungal skin infections. We analyzed the mycobiome of mycelia-like skin crusts of the yellow-throated bunting (Emberiza elegans), a common migratory songbird with declining population in Korea, using DNA metabarcoding targeting the internal transcribed spacer 1 (ITS1) region, the actin (ACT) gene, and the translation elongation factor 1 - α (TEF) gene. Our analysis revealed that Cladosporium was the predominant genus (~ 60% sequence reads) in fungal mycelia-like tissues on the skins of yellow-throated buntings and detected a large number of DNA sequences similar to those of species belonging to the Cladosporium cladosporioides species complex. This is the first study to report possible infection in buntings by Cladosporium, including species known to infect humans and other animals. Further research on the causal relationship between birds and fungi is needed for pathogen management and conservation of Asian songbirds along the migration flyway.