Geographical distribution of the Cryptococcus gattii species complex: a systematic review.

The taxonomy of the Cryptococcus gattii species complex continues to evolve, and has been divided into five pathogenic species. The objective of this systematic review was to summarise the geographical distribution of the C gattii species complex and the species within the C gattii species complex. We searched PubMed for articles related to human, animal, ecological, or laboratory-based studies of C gattii species complex isolates with traceable geographical origin published from January, 1970, until September, 2021. Having extracted their geographical origin, we used ArcMap to construct maps according to the highest degree of resolution allowed by their reported taxonomy, to reflect the most likely area of transmission on the basis of published reports of human isolates. 604 such articles were included in the study. This review indicated that although C gattii species complex isolates have been reported globally, understanding their heterogeneous geographical distribution by species can have implications for researchers and clinicians in formulating research questions and considering diagnostic quandaries.

Susceptibility Testing of Environmental and Clinical Aspergillus sydowii Demonstrates Potent Activity of Various Antifungals.

The genus Aspergillus consists of a vast number of medically and environmentally relevant species. Aspergillus species classified in series Versicolores are ubiquitous in the environment and include the opportunistic pathogen Aspergillus sydowii, which is associated with onychomycosis and superficial skin infections. Despite frequent clinical reports of A. sydowii and related series Versicolores species, antifungal susceptibility data are scarce, hampering optimal treatment choices and subsequent patient outcomes. Here, we employed antifungal susceptibility testing (AFST) based on microbroth dilution on a set of 155 series Versicolores strains using the common antifungals amphotericin B, itraconazole, voriconazole, posaconazole, isavuconazole and micafungin with the addition of luliconazole and olorofim. All strains were identified using partial calmodulin gene sequencing, with 145 being A. sydowii, seven A. creber and three A. versicolor, using the latest taxonomic insights. Overall, tested antifungals were potent against the entire strain collection. In comparison to A. fumigatus, azole and amphotericin B MICs were slightly elevated for some strains. AFST with luliconazole and olorofim, here reported for the first time, displayed the highest in vitro activity, making these antifungals interesting alternative drugs but clinical studies are warranted for future therapeutic use.

Clinical response to prednisone in a severe case of infant-juvenile paracoccidioidomycosis.

BACKGROUND: Paracoccidioidomycosis is a neglected tropical disease caused by fungi of the genus Paracoccidioides. A wide range of symptoms is related to the disease; however, lungs and skin are the sites predominantly affected. The disease is mostly seen in people living in rural areas in Latin America. CASE REPORT: We present a pediatric case of severe disseminated paracoccidioidomycosis that slowly responded to the antifungal treatment. Within three months, symptoms evolved into hepatosplenomegaly, necrotic cervical and abdominal lymph nodes, and splenic abscess. Clinical response to amphotericin B deoxycholate and itraconazole was slow, resulting in pleural and peritoneal cavity effusions, heart failure and shock. Amphotericin B deoxycholate was replaced by the liposomal formulation, with no response. Subsequently, prednisone was added to the treatment, which led to improvement in the clinical response. Serological Paracoccidioides antibody titers were atypical, with very low titers in the critical phase and significant increase during the convalescence phase. The infection was finally cleared up with amphotericin B deoxycholate, liposomal amphotericin B and the use of corticosteroids. Paracoccidioidomycosis serology was non-reactive two years post-discharge. CONCLUSIONS: Due to the intense inflammatory response triggered by Paracoccidioides cells, giving low-dose prednisone for a short period of time modulated the inflammatory response and supported antifungal treatment.

Fusarioid keratitis and other superficial infections: A 10-years prospective study from Northeastern Brazil.

BACKGROUND: Fusarium and allied genera (fusarioid) species are common colonizers of roots and aerial plant parts, or act as phytopathogens in forestry and horticultural or grain crops. However, they can also cause a wide range of infections in humans, including onychomycosis, cutaneous and invasive infections. Fusarioid keratitis is characterized by an infection of the cornea with a suppurative and ulcerative appearance, which may cause damage to vision and permanent blindness. The aim of the present study was to investigate the prevalence of fusarioid species, biofilm formation and antifungal susceptibility profiling of clinical isolates recovered from patients with keratitis and dermatomycoses. METHODOLOGY/PRINCIPAL FINDINGS: The study was performed between March, 2012-December, 2022. Demographic, clinical and epidemiological data of patients were also collected. In the present study, most of the patients with keratitis were male (74%), had a median age of 42 years old, worked with plant material or debris and 26% of them reported eye trauma. Regarding dermatomycosis, most of patients were female and exhibited toenail lesions. Forty-seven isolates belonged to the genus Neocosmospora (78.33%), nine to the Fusarium fujikuroi (15%) and four to the Fusarium oxysporum (6.66%) species complexes. Several strains were moderate biofilm producers, specifically among Fusarium annulatum. Most strains showed increased MICs to amphotericin B and ketoconazole and low MICs to itraconazole. MICs ranged from 0.25 to 16 µg/mL for amphotericin B, 0.0625 to >16 µg/mL for ketoconazole and 0.125 to 8 for itraconazole. CONCLUSIONS/SIGNIFICANCE: It is possible to conclude that fusarioid keratitis in Northeastern Brazil is an important and neglected disease, given the high number of cases, increased need for keratoplasty and poor outcome of the disease.

Improving disseminated histoplasmosis diagnosis in HIV/AIDS patients in Suriname: The role of a urine lateral flow assay.

Histoplasmosis is a frequent cause of infections in people living with HIV/AIDS (PLWHA). This study introduces the application of a Histoplasma capsulatum urine antigen lateral flow assay (LFA) for diagnosing disseminated histoplasmosis in PLWHA in Suriname. The LFA's diagnostic accuracy was compared with the current diagnostic approach, aiming to assess whether this test resulted in improved early detection and management. Additionally, the prevalence of histoplasmosis among advanced stage HIV patients without clinical suspicion of infection was evaluated using the same LFA. In total, 98 patients were included in the study, of which 58 were classified as "possible disseminated histoplasmosis (DH)" based on clinical criteria and 40 as "controls". Of these possible DH cases, only 19 (32.7%) had a positive LFA. During the study, decisions for treatment were made without the treating physician being aware of the LFA result. Only 55% of the patients who started treatment for histoplasmosis based on clinical criteria had a positive LFA, and 21% of untreated patients had a positive LFA. This study shows that combining clinical signs with LFA results enhances diagnostic accuracy and is cost effective, resulting in better treatment decisions.

Unveiling Trichosporon austroamericanum sp. nov.: A Novel Emerging Opportunistic Basidiomycetous Yeast Species.

During an epidemiological survey, a potential novel species within the basidiomycetous yeast genus Trichosporon was observed. The clinical strain was obtained from a urine sample taken from a Brazilian kidney transplant recipient. The strain was molecularly identified using the intergenic spacer (IGS1) ribosomal DNA locus and a subsequent phylogenetic analysis showed that multiple strains that were previously reported by other studies shared an identical IGS1-genotype most closely related to that of Trichosporon inkin. However, none of these studies provided an in-depth characterization of the involved strains to describe it as a new taxon. Here, we present the novel clinically relevant yeast for which we propose the name Trichosporon austroamericanum sp. nov. (holotype CBS H-24937). T. austroamericanum can be distinguished from other siblings in the genus Trichosporon using morphological, physiological, and phylogenetic characters.

Aspergillus hubkae, a Novel Species Isolated from a Patient with Probable Invasive Pulmonary Aspergillosis.

A 50-year-old man, previously diagnosed with pulmonary tuberculosis and lung cavities, presented with symptoms including fever, shortness of breath, and cough. A pulmonary CT scan revealed multiple cavities, consolidation and tree-in-bud in the upper lungs. Further investigation through direct examination of bronchoalveolar lavage fluid showed septate hyphae with dichotomous acute branching. Subsequent isolation and morphological analysis identified the fungus as belonging to Aspergillus section Nigri. The patient was diagnosed with probable invasive pulmonary aspergillosis and successfully treated with a three-month oral voriconazole therapy. Phylogenetic analysis based on partial ß-tubulin, calmodulin and RNA polymerase second largest subunit sequences revealed that the isolate represents a putative new species related to Aspergillus brasiliensis, and is named Aspergillus hubkae here. Antifungal susceptibility testing demonstrated that the isolate is resistant to itraconazole but susceptible to voriconazole. This phenotypic and genetic characterization of A. hubkae, along with the associated case report, will serve as a valuable resource for future diagnoses of infections caused by this species. It will also contribute to more precise and effective patient management strategies in similar clinical scenarios.

Histoplasmosis: An epidemiological and clinical update in China, review and a case report.

Histoplasmosis is a systemic mycosis caused by the dimorphic fungus in the genus Histoplasma. Histoplasmosis is overlooked in China. This study aims to provide an epidemiological and clinical update on histoplasmosis in China by literature review. We reviewed cases of histoplasmosis reported in recent 11 years and described a case of histoplasmosis-triggered hemophagocytic lymphohistiocytosis (HLH) in an immunocompetent patient. A total of 225 cases of histoplasmosis diagnosed in China between 2012 and 2022 were involved in this study, compared with 300 cases reviewed from 1990 to 2011, an increasing number of cases of histoplasmosis have been diagnosed in the last 11 years. The majority of cases of histoplasmosis were autochthonous cases, mainly from provinces Sichuan (56/225, 24.9%), Hunan (50/225, 22.2%), Guangdong (31/225, 13.8%), and Yunnan (24/225, 10.7%). Higher incidence (52.5%, 53/99) of histoplasmosis occurred in immunocompetent patients which is similar to those from the previous 21 years, and the prevalence of the disease did not vary highly over time. Of note, the number of histoplasmosis cases is increasing, and the geographic distribution is shifting southwards over time. Improved awareness is critically important for informing clinical practice in China.

Global guideline for the diagnosis and management of cryptococcosis: an initiative of the ECMM and ISHAM in cooperation with the ASM.

Cryptococcosis is a major worldwide disseminated invasive fungal infection. Cryptococcosis, particularly in its most lethal manifestation of cryptococcal meningitis, accounts for substantial mortality and morbidity. The breadth of the clinical cryptococcosis syndromes, the different patient types at-risk and affected, and the vastly disparate resource settings where clinicians practice pose a complex array of challenges. Expert contributors from diverse regions of the world have collated data, reviewed the evidence, and provided insightful guideline recommendations for health practitioners across the globe. This guideline offers updated practical guidance and implementable recommendations on the clinical approaches, screening, diagnosis, management, and follow-up care of a patient with cryptococcosis and serves as a comprehensive synthesis of current evidence on cryptococcosis. This Review seeks to facilitate optimal clinical decision making on cryptococcosis and addresses the myriad of clinical complications by incorporating data from historical and contemporary clinical trials. This guideline is grounded on a set of core management principles, while acknowledging the practical challenges of antifungal access and resource limitations faced by many clinicians and patients. More than 70 societies internationally have endorsed the content, structure, evidence, recommendation, and pragmatic wisdom of this global cryptococcosis guideline to inform clinicians about the past, present, and future of care for a patient with cryptococcosis.

Complete Genome Sequence of the Itraconazole Decreased Susceptible Madurella fahalii Type-Strain CBS 129176.

Madurella fahalii is a causative agent of the implantation mycosis mycetoma with decreased susceptibility to itraconazole, the preferred therapeutic drug to combat mycetoma. Here, we report the M. fahalii type-strain CBS 129176 genome assembly and annotation to identify a glutamic acid insert near the azole-binding pocket in the Cyp51A protein.

A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis.

BACKGROUND: In-house real-time PCR (qPCR) is increasingly used to diagnose the so-called endemic mycoses as commercial assays are not widely available. OBJECTIVES: To compare the performance of different molecular diagnostic assays for detecting Histoplasma capsulatum and Coccidioides spp. in five European reference laboratories. METHODS: Two blinded external quality assessment (EQA) panels were sent to each laboratory that performed the analysis with their in-house assays. Both panels included a range of concentrations of H. capsulatum (n = 7) and Coccidioides spp. (n = 6), negative control and DNA from other fungi. Four laboratories used specific qPCRs, and one laboratory a broad-range fungal conventional PCR (cPCR) and a specific cPCR for H. capsulatum with subsequent sequencing. RESULTS: qPCR assays were the most sensitive for the detection of H. capsulatum DNA. The lowest amount of H. capsulatum DNA detected was 1-4 fg, 0.1 pg and 10 pg for qPCRs, specific cPCR and broad-range cPCR, respectively. False positive results occurred with high concentrations of Blastomyces dermatitidis DNA in two laboratories and with Emergomyces spp. in one laboratory. For the Coccidioides panel, the lowest amount of DNA detected was 1-16 fg by qPCRs and 10 pg with the broad-range cPCR. One laboratory reported a false positive result by qPCR with high load of Uncinocarpus DNA. CONCLUSION: All five laboratories were able to correctly detect H. capsulatum and Coccidioides spp. DNA and qPCRs had a better performance than specific cPCR and broad-range cPCR. EQAs may help standardise in-house molecular tests for the so-called endemic mycoses improving patient management.

Whole-genome sequencing confirms a persistent candidaemia clonal outbreak due to multidrug-resistant Candida parapsilosis.

OBJECTIVES: Although perceived as a rare clinical entity, recent studies have noted the emergence of MDR C. parapsilosis (MDR-Cp) isolates from single patients (resistant to both azole and echinocandins). We previously reported a case series of MDR-Cp isolates carrying a novel FKS1R658G mutation. Herein, we identified an echinocandin-naive patient infected with MDR-Cp a few months after the previously described isolates. WGS and CRISPR-Cas9 editing were used to explore the origin of the new MDR-Cp isolates, and to determine if the novel mutation confers echinocandin resistance. METHODS: WGS was applied to assess the clonality of these isolates and CRISPR-Cas9 editing and a Galleria mellonella model were used to examine whether FKS1R658G confers echinocandin resistance. RESULTS: Fluconazole treatment failed, and the patient was successfully treated with liposomal amphotericin B (LAMB). WGS proved that all historical and novel MDR-Cp strains were clonal and distant from the fluconazole-resistant outbreak cluster in the same hospital. CRISPR-Cas9 editing and G. mellonella virulence assays confirmed that FKS1R658G confers echinocandin resistance in vitro and in vivo. Interestingly, the FKS1R658G mutant showed a very modest fitness cost compared with the parental WT strain, consistent with the persistence of the MDR-Cp cluster in our hospital. CONCLUSIONS: Our study showcases the emergence of MDR-Cp isolates as a novel threat in clinical settings, which undermines the efficacy of the two most widely used antifungal drugs against candidiasis, leaving only LAMB as a last resort. Additionally, surveillance studies and WGS are warranted to effectively establish infection control and antifungal stewardship strategies.

Candida parapsilosis isolates carrying mutations outside FKS1 hotspot regions confer high echinocandin tolerance and facilitate the development of echinocandin resistance.

Candida parapsilosis is a significant cause of candidemia worldwide. Echinocandin-resistant (ECR) and echinocandin-tolerant (ECT) C. parapsilosis isolates have been reported in various countries but are rare. Resistance and tolerance are predominantly caused by mutations related to the hotspot (HS) regions of the FKS1 gene. A relatively high proportion of clinical C. parapsilosis isolates carrying mutations outside the HS regions has been noted in some studies, but an association with echinocandin (EC) resistance or tolerance was not explored. Herein, CRISPR-Cas9 was used and the association between amino acid substitution in FKS1 outside HS 1/2 (V595I, S745L, M1328I, F1386S, and A1422G) with EC susceptibility profile was delineated. None of the mutations conferred EC resistance, but they resulted in a significantly higher level of EC tolerance than the parental isolate, ATCC 22019. When incubated on agar plates containing ECs, specifically caspofungin and micafungin, ECR colonies were exclusively observed among ECT isolates, particularly mutants carrying V595I, S745L, and F1386S. Additionally, mutants had significantly better growth rates in yeast extract peptone dextrose (YPD) and YPD containing agents inducing membrane and oxidative stresses. The mutants had a trivial fitness cost in the Galleria mellonella model relative to ATCC 22019. Collectively, this study supports epidemiological studies to catalog mutations occurring outside the HS regions of FKS1, even if they do not confer EC resistance. These mutations are important as they potentially confer a higher level of EC tolerance and a higher propensity to develop EC resistance, therefore unveiling a novel mechanism of EC tolerance in C. parapsilosis. The identification of EC tolerance in C. parapsilosis may have direct clinical benefit in patient management.

Comparing genomic variant identification protocols for Candida auris.

Genomic analyses are widely applied to epidemiological, population genetic and experimental studies of pathogenic fungi. A wide range of methods are employed to carry out these analyses, typically without including controls that gauge the accuracy of variant prediction. The importance of tracking outbreaks at a global scale has raised the urgency of establishing high-accuracy pipelines that generate consistent results between research groups. To evaluate currently employed methods for whole-genome variant detection and elaborate best practices for fungal pathogens, we compared how 14 independent variant calling pipelines performed across 35 Candida auris isolates from 4 distinct clades and evaluated the performance of variant calling, single-nucleotide polymorphism (SNP) counts and phylogenetic inference results. Although these pipelines used different variant callers and filtering criteria, we found high overall agreement of SNPs from each pipeline. This concordance correlated with site quality, as SNPs discovered by a few pipelines tended to show lower mapping quality scores and depth of coverage than those recovered by all pipelines. We observed that the major differences between pipelines were due to variation in read trimming strategies, SNP calling methods and parameters, and downstream filtration criteria. We calculated specificity and sensitivity for each pipeline by aligning three isolates with chromosomal level assemblies and found that the GATK-based pipelines were well balanced between these metrics. Selection of trimming methods had a greater impact on SAMtools-based pipelines than those using GATK. Phylogenetic trees inferred by each pipeline showed high consistency at the clade level, but there was more variability between isolates from a single outbreak, with pipelines that used more stringent cutoffs having lower resolution. This project generated two truth datasets useful for routine benchmarking of C. auris variant calling, a consensus VCF of genotypes discovered by 10 or more pipelines across these 35 diverse isolates and variants for 2 samples identified from whole-genome alignments. This study provides a foundation for evaluating SNP calling pipelines and developing best practices for future fungal genomic studies.

Identification of Clinical Trichosporon asteroides Strains by MALDI-TOF Mass Spectrometry: Evaluation of the Bruker Daltonics Commercial System and an In-House Developed Library.

Trichosporon asteroides is an emerging yeast-like pathogen commonly misidentified by commercial biochemical identification systems. We evaluated the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of 21 clinical T. asteroides strains using the Bruker Daltonics database (BDAL) and an in-house developed library. Mass spectra were obtained by the FlexControl system v.3.4, and characterizations were performed in the Biotyper BDAL database v.4.1 and the developed in-house library. Species identification for T. asteroides failed as all 21 strains were misidentified as T. japonicum (log-scores 1.89-2.19). Extending the existing database was crucial to achieving 100% correct species-level identification and accurate distinction between species. Our results indicate that the commercial BDAL database has no discriminatory power to distinguish between T. japonicum and T. asteroides. Whereas improvement of the current BDAL database is pending, we strongly advise system users not to exclude the possibility of the failure to report T. asteroides.

High-Throughput Microsatellite Markers Development for Genetic Characterization of Emerging Sporothrix Species.

Sporotrichosis is the main subcutaneous mycosis worldwide transmitted by animal or plant vectors and often escalates to outbreaks or epidemics. The current cat-transmitted sporotrichosis driven by Sporothrix brasiliensis has become a significant public health issue in South America. Transmission dynamics remain enigmatic due to the lack of development of polymorphic markers for molecular epidemiological analysis. This study used a high-throughput mining strategy to characterize simple sequence repeat (SSR) markers from Sporothrix genomes. A total of 118,140-143,912 SSR loci were identified (82,841-98,369 unique markers), with a 3651.55-3804.65 SSR/Mb density and a majority of dinucleotides motifs (GC/CG). We developed a panel of 15 highly polymorphic SSR markers suitable for genotyping S. brasiliensis, S. schenckii, and S. globosa. PCR amplification revealed 240 alleles in 180 Sporothrix isolates with excellent polymorphic information content (PIC = 0.9101), expected heterozygosity (H = 0.9159), and discriminating power (D = 0.7127), supporting the effectiveness of SSR markers in uncovering cryptic genetic diversity. A systematic population genetic study estimated three clusters, corresponding to S. brasiliensis (population 1, n = 97), S. schenckii (population 2, n = 49), and S. globosa (population 3, n = 34), with a weak signature of mixed ancestry between populations 1 and 2 or 3 and 2. Partitioning of genetic variation via AMOVA revealed highly structured populations (ΦPT = 0.539; Nm = 0.213; p < 0.0001), with approximately equivalent genetic variability within (46%) and between (54%) populations. Analysis of SSR diversity supports Rio de Janeiro (RJ) as the center of origin for contemporary S. brasiliensis infections. The recent emergence of cat-transmitted sporotrichosis in northeastern Brazil indicates an RJ-Northeast migration resulting in founder effects during the introduction of diseased animals into sporotrichosis-free areas. Our results demonstrated high cross-species transferability, reproducibility, and informativeness of SSR genetic markers, helping dissect deep and fine-scale genetic structures and guiding decision making to mitigate the harmful effects of the expansion of cat-transmitted sporotrichosis.