Genome assemblies of two rare opportunistic yeast pathogens: Diutina rugosa (syn. Candida rugosa) and Trichomonascus ciferrii (syn. Candida ciferrii)

Infections caused by opportunistic yeast pathogens have increased over the last years. These infections can be originated by a large number of diverse yeast species of varying incidence, and with distinct clinically relevant phenotypic traits, such as different susceptibility profiles to antifungal drugs, which challenge diagnosis and treatment. Diutina rugosa (syn. Candida rugosa) and Trichomonascus ciferrii (syn. Candida ciferrii) are two opportunistic rare yeast pathogens, which low incidence (< 1%) limits available clinical experience. Furthermore, these yeasts have elevated Minimum Inhibitory Concentration (MIC) levels to at least one class of antifungal agents. This makes it more difficult to manage their infections, and thus they are associated with high rates of mortality and clinical failure. With the aim of improving our knowledge on these opportunistic pathogens, we assembled and annotated their genomes. A phylogenomics approach revealed that genes specifically duplicated in each of the two species are often involved in transmembrane transport activities. These genomes and the reconstructed complete catalog of gene phylogenies and homology relationships constitute useful resources for future studies on these pathogens.This work received funding from the European Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie Grant Agreement No. H2020-MSCA-ITN-2014-642095. TG group also acknowledges support from the Spanish Ministry of Economy, Industry, and Competitiveness (MEIC) for the EMBL partnership, and grants ‘Centro de Excelencia Severo Ochoa 2013–2017’ SEV-2012-0208, and BFU2015-67107 co-founded by European Regional Development Fund (ERDF); from the CERCA Program/Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR857, and grants from the European Union’s Horizon 2020 Research and Innovation Program under the Grant Agreements No. ERC-2016-724173, and MSCA-747607. TG also receives support from an INB grant (PT17/0009/0023 – ISCIII-SGEFI/ERDF). CLF received funding from Christian Doppler Laboratory for Fungal Infections: Avoid, find, and treat! The authors thank all Gabaldón lab members for helpful discussions on this study, specially Marina Marcet-Houben

Efficacy of LAMB against Emerging Azole- and Multidrug-Resistant Candida parapsilosis Isolates in the Galleria mellonella Model

While being the third leading cause of candidemia worldwide, numerous studies have shown severe clonal outbreaks due to fluconazole-resistant (FLCR) Candida parapsilosis isolates associated with fluconazole therapeutic failure (FTF) with enhanced mortality. More recently, multidrug resistant (MDR) C. parapsilosis blood isolates have also been identified that are resistant to both azole and echinocandin drugs. Amphotericin B (AMB) resistance is rarely reported among C. parapsilosis isolates and proper management of bloodstream infections due to FLZR and MDR isolates requires prompt action at the time of outbreak. Therefore, using a well-established Galleria mellonella model, we assessed whether (a) laboratory-based findings on azole or echinocandin (micafungin) resistance in C. parapsilosis lead to therapeutic failure, (b) LAMB could serve as an efficient salvage treatment option, and (c) distinct mutations in ERG11 impact mortality. Our in vivo data confirm fluconazole inefficacy against FLCR C. parapsilosis isolates carrying Y132F, Y132F + K143R, Y132F + G307A, and G307A + G458S in Erg11p, while LAMB proved to be an efficacious accessible option against both FLCR and MDR C. parapsilosis isolates. Moreover, positive correlation of in vitro and in vivo data further highlights the utility of G. melonella as a reliable model to investigate azole and polyene drug efficacy.Christian DopplerForschungsgesellschaft (CD-Labor Invasive Pilzinfektionen)This work was financially supported by Christian DopplerForschungsgesellschaft (CD-Labor Invasive Pilzinfektionen) to C.L.-F

Antifungal susceptibility testing in Candida, Aspergillus and Cryptococcus infections: are the MICs Useful for clinicians?

Invasive fungal infections (IFI) represent a global issue and affect various patient populations. In recent years, resistant fungal isolates showing increased MICs to azoles or echinocandins have been reported, and their potential clinical impact has been investigated

Whole-genome sequencing of the opportunistic yeast pathogen candida inconspicua uncovers its hybrid origin

Fungal infections such as those caused by Candida species are increasingly common complications in immunocompromised patients. The list of causative agents of candidiasis is growing and comprises a set of emerging species whose relative global incidence is rare but recurrent. This is the case of Candida inconspicua, which prevalence has increased 10-fold over the last years. To gain novel insights into the emergence of this opportunistic pathogen and its genetic diversity, we performed whole genome sequencing of the type strain (CBS180), and of 10 other clinical isolates. Our results revealed high levels of genetic heterozygosity structured in non-homogeneous patterns, which are indicative of a hybrid genome shaped by events of loss of heterozygosity (LOH). All analyzed strains were hybrids and could be clustered into two distinct clades. We found large variability across strains in terms of ploidy, patterns of LOH, and mitochondrial genome heterogeneity that suggest potential admixture between hybrids. Altogether, our results identify a new hybrid species with virulence potential toward humans and underscore the potential role of hybridization in the emergence of novel pathogenic lineages.This work received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Grant Agreement No. H2020-MSCA-ITN-2014-642095. TG group also acknowledges support from the Spanish Ministry of Economy, Industry, and Competitiveness (MEIC) for the EMBL partnership, and grants ‘Centro de Excelencia Severo Ochoa 2013–2017’ SEV-2012-0208, and BFU2015-67107 co-founded by European Regional Development Fund (ERDF); from the CERCA Programme/Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR857, and grants from the European Union’s Horizon 2020 Research and Innovation Programme under the Grant Agreement No. ERC-2016-724173. TG also receives support from a INB grant (PT17/0009/0023 – ISCIII-SGEFI/ERDF

Clinical evaluation of a Mucorales-specific real-time PCR assay in tissue and serum samples

Molecular diagnostic assays can accelerate the diagnosis of fungal infections and subsequently improve patient outcomes. In particular, the detection of infections due to Mucorales is still challenging for laboratories and physicians. The aim of this study was to evaluate a probe-based Mucorales-specific real-time PCR assay (Muc18S) using tissue and serum samples from patients suffering from invasive mucormycosis (IMM). This assay can detect a broad range of clinically relevant Mucorales species and can be used to complement existing diagnostic tests or to screen high-risk patients. An advantage of the Muc18S assay is that it exclusively detects Mucorales species allowing the diagnosis of Mucorales DNA without sequencing within a few hours. In paraffin-embedded tissue samples this PCR-based method allowed rapid identification of Mucorales in comparison with standard methods and showed 91 % sensitivity in the IMM tissue samples. We also evaluated serum samples, an easily accessible material, from patients at risk from IMM. Mucorales DNA was detected in all patients with probable/proven IMM (100 %) and in 29 % of the possible cases. Detection of IMM in serum could enable an earlier diagnosis (up to 21 days) than current methods including tissue samples, which were gained mainly post-mortem. A screening strategy for high-risk patients, which would enable targeted treatment to improve patient outcomes, is therefore possible

High-frequency triazole resistance found in nonculturable aspergillus fumigatus from lungs of patients with chronic fungal disease

Background. Oral triazole therapy is well established for the treatment of invasive (IPA), allergic (ABPA), and chronic pulmonary (CPA) aspergillosis, and is often long-term. Triazole resistance rates are rising internationally. Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequency of triazole resistance. Methods. Using an ultrasensitive real-time polymerase chain reaction (PCR) assay for Aspergillus spp., we assessed respiratory fungal load in bronchoalveolar lavage (BAL) and sputum specimens. In a subset of PCR-positive, culture negative samples, we further amplified the CYP51A gene to detect key single-nucleotide polymorphisms (SNPs) associated with triazole resistance. Results. Aspergillus DNA was detected in BAL from normal volunteers (4/11, 36.4%) and patients with culture or microscopy confirmed IPA (21/22, 95%). Aspergillus DNA was detected in sputum in 15 of 19 (78.9%) and 30 of 42 (71.4%) patients with ABPA and CPA, compared with 0% and 16.7% by culture, respectively. In culture-negative, PCR-positive samples, we detected triazole-resistance mutations (L98H with tandem repeat [TR] and M220) within the drug target CYP51A in 55.1% of samples. Six of 8 (75%) of those with ABPA and 12 of 24 (50%) with CPA had resistance markers present, some without prior triazole treatment, and in most despite adequate plasma drug concentrations around the time of sampling. Conclusions. The very low organism burdens of fungi causing infection have previously prevented direct culture and detection of antifungal resistance in clinical samples. These findings have major implications for the sustainability of triazoles for human antifungal therapy

Phylogenetic diversity of human pathogenic Fusarium and emergence of uncommon virulent species

OBJECTIVES: Fusarium species cause a broad spectrum of infections. However, little is known about the etiological agents to the species level. We identified Fusarium species isolated from clinical specimens including those of high risk patients to better understand the species involved in the pathogenesis. METHODS: A set of 44 Fusarium isolates were identified by two-locus sequence typing using partial sequences of the second largest subunit of RNA polymerase (RPB2) and translation elongation factor 1 alpha (TEF-1α). RESULTS: The identified species belonged to four species complexes (SC); the most common SC was Fusarium solani (FSSC) (75%), followed by Fusarium oxysporum (FOSC) (4.5%), Fusarium fujikuroi (FFSC) (13.6%), and Fusarium dimerum (FDSC) (6.8%). Sites of infections were nails (n = 19, 43.2%), skin (n = 7, 15.9%), cornea (n = 6, 13.6%), blood (n = 3, 9%), wound (n = 4, 6.8%), burn (n = 2, 4.5%), tissue (n = 2, 4.5%), and urine (n = 1, 2.27%). Fusarium acutatum was rare and seem restricted to the Middle East. Comorbidities associated with invasive infections were hematological malignancy and autoimmune disorders. CONCLUSIONS: Members of the FSSC predominantly caused cornea, nail and bloodstream infections. Less frequently encountered were the FOSC, FFSC and FDSC. More accurate molecular identification of Fusarium species is important to predict therapeutic outcome and the emergence of these species

The Antifungal Pipeline: Fosmanogepix, Ibrexafungerp, Olorofim, Opelconazole, and Rezafungin

The epidemiology of invasive fungal infections is changing, with new populations at risk and the emergence of resistance caused by the selective pressure from increased usage of antifungal agents in prophylaxis, empiric therapy, and agriculture. Limited antifungal therapeutic options are further challenged by drug-drug interactions, toxicity, and constraints in administration routes. Despite the need for more antifungal drug options, no new classes of antifungal drugs have become available over the last 2 decades, and only one single new agent from a known antifungal class has been approved in the last decade. Nevertheless, there is hope on the horizon, with a number of new antifungal classes in late-stage clinical development. In this review, we describe the mechanisms of drug resistance employed by fungi and extensively discuss the most promising drugs in development, including fosmanogepix (a novel Gwt1 enzyme inhibitor), ibrexafungerp (a first-in-class triterpenoid), olorofim (a novel dihyroorotate dehydrogenase enzyme inhibitor), opelconazole (a novel triazole optimized for inhalation), and rezafungin (an echinocandin designed to be dosed once weekly). We focus on the mechanism of action and pharmacokinetics, as well as the spectrum of activity and stages of clinical development. We also highlight the potential future role of these drugs and unmet needs

Taskforce report on the diagnosis and clinical management of COVID-19 associated pulmonary aspergillosis

none28siPurpose: Invasive pulmonary aspergillosis (IPA) is increasingly reported in patients with severe coronavirus disease 2019 (COVID-19) admitted to the intensive care unit (ICU). Diagnosis and management of COVID-19 associated pulmonary aspergillosis (CAPA) are challenging and our aim was to develop practical guidance. Methods: A group of 28 international experts reviewed current insights in the epidemiology, diagnosis and management of CAPA and developed recommendations using GRADE methodology. Results: The prevalence of CAPA varied between 0 and 33%, which may be partly due to variable case definitions, but likely represents true variation. Bronchoscopy and bronchoalveolar lavage (BAL) remain the cornerstone of CAPA diagnosis, allowing for diagnosis of invasive Aspergillus tracheobronchitis and collection of the best validated specimen for Aspergillus diagnostics. Most patients diagnosed with CAPA lack traditional host factors, but pre-existing structural lung disease and immunomodulating therapy may predispose to CAPA risk. Computed tomography seems to be of limited value to rule CAPA in or out, and serum biomarkers are negative in 85% of patients. As the mortality of CAPA is around 50%, antifungal therapy is recommended for BAL positive patients, but the decision to treat depends on the patients’ clinical condition and the institutional incidence of CAPA. We recommend against routinely stopping concomitant corticosteroid or IL-6 blocking therapy in CAPA patients. Conclusion: CAPA is a complex disease involving a continuum of respiratory colonization, tissue invasion and angioinvasive disease. Knowledge gaps including true epidemiology, optimal diagnostic work-up, management strategies and role of host-directed therapy require further study.openVerweij P.E.; Bruggemann R.J.M.; Azoulay E.; Bassetti M.; Blot S.; Buil J.B.; Calandra T.; Chiller T.; Clancy C.J.; Cornely O.A.; Depuydt P.; Koehler P.; Lagrou K.; de Lange D.; Lass-Florl C.; Lewis R.E.; Lortholary O.; Liu P.-W.L.; Maertens J.; Nguyen M.H.; Patterson T.F.; Rijnders B.J.A.; Rodriguez A.; Rogers T.R.; Schouten J.A.; Wauters J.; van de Veerdonk F.L.; Martin-Loeches I.Verweij P.E.; Bruggemann R.J.M.; Azoulay E.; Bassetti M.; Blot S.; Buil J.B.; Calandra T.; Chiller T.; Clancy C.J.; Cornely O.A.; Depuydt P.; Koehler P.; Lagrou K.; de Lange D.; Lass-Florl C.; Lewis R.E.; Lortholary O.; Liu P.-W.L.; Maertens J.; Nguyen M.H.; Patterson T.F.; Rijnders B.J.A.; Rodriguez A.; Rogers T.R.; Schouten J.A.; Wauters J.; van de Veerdonk F.L.; Martin-Loeches I