Scedosporiosis and lomentosporiosis: modern perspectives on these difficult-to-treat rare mold infections.

SUMMARYAlthough Scedosporium species and Lomentospora prolificans are uncommon causes of invasive fungal diseases (IFDs), these infections are associated with high mortality and are costly to treat with a limited armamentarium of antifungal drugs. In light of recent advances, including in the area of new antifungals, the present review provides a timely and updated overview of these IFDs, with a focus on the taxonomy, clinical epidemiology, pathogenesis and host immune response, disease manifestations, diagnosis, antifungal susceptibility, and treatment. An expansion of hosts at risk for these difficult-to-treat infections has emerged over the last two decades given the increased use of, and broader population treated with, immunomodulatory and targeted molecular agents as well as wider adoption of antifungal prophylaxis. Clinical presentations differ not only between genera but also across the different Scedosporium species. L. prolificans is intrinsically resistant to most currently available antifungal agents, and the prognosis of immunocompromised patients with lomentosporiosis is poor. Development of, and improved access to, diagnostic modalities for early detection of these rare mold infections is paramount for timely targeted antifungal therapy and surgery if indicated. New antifungal agents (e.g., olorofim, fosmanogepix) with novel mechanisms of action and less cross-resistance to existing classes, availability of formulations for oral administration, and fewer drug-drug interactions are now in late-stage clinical trials, and soon, could extend options to treat scedosporiosis/lomentosporiosis. Much work remains to increase our understanding of these infections, especially in the pediatric setting. Knowledge gaps for future research are highlighted in the review.

Histoplasmosis: A systematic review to inform the World Health Organization of a fungal priority pathogens list.

Histoplasmosis, a significant mycosis primarily prevalent in Africa, North and South America, with emerging reports globally, poses notable health challenges, particularly in immunocompromised individuals such as people living with HIV/AIDS and organ transplant recipients. This systematic review, aimed at informing the World Health Organization's Fungal Priority Pathogens List, critically examines literature from 2011 to 2021 using PubMed and Web of Science, focusing on the incidence, mortality, morbidity, antifungal resistance, preventability, and distribution of Histoplasma. We also found a high prevalence (22%-44%) in people living with HIV, with mortality rates ranging from 21% to 53%. Despite limited data, the prevalence of histoplasmosis seems stable, with lower estimates in Europe. Complications such as central nervous system disease, pulmonary issues, and lymphoedema due to granuloma or sclerosis are noted, though their burden remains uncertain. Antifungal susceptibility varies, particularly against fluconazole (MIC: ≥32 mg/l) and caspofungin (MICs: 4-32 mg/l), while resistance to amphotericin B (MIC: 0.125-0.16 mg/l), itraconazole (MICs: 0.004-0.125 mg/l), and voriconazole (MICs: 0.004-0.125 mg/l) remains low. This review identifies critical knowledge gaps, underlining the need for robust, globally representative surveillance systems to better understand and combat this fungal threat.

Fusarium species,Scedosporium species, and Lomentospora prolificans: A systematic review to inform the World Health Organization priority list of fungal pathogens.

Recognizing the growing global burden of fungal infections, the World Health Organization established a process to develop a priority list of fungal pathogens (FPPL). In this systematic review, we aimed to evaluate the epidemiology and impact of infections caused by Fusarium spp., Scedosporium spp., and Lomentospora prolificans to inform the first FPPL. PubMed and Web of Sciences databases were searched to identify studies published between January 1, 2011 and February 23, 2021, reporting on mortality, complications and sequelae, antifungal susceptibility, preventability, annual incidence, and trends. Overall, 20, 11, and 9 articles were included for Fusarium spp., Scedosporium spp., and L. prolificans, respectively. Mortality rates were high in those with invasive fusariosis, scedosporiosis, and lomentosporiosis (42.9%-66.7%, 42.4%-46.9%, and 50.0%-71.4%, respectively). Antifungal susceptibility data, based on small isolate numbers, showed high minimum inhibitory concentrations (MIC)/minimum effective concentrations for most currently available antifungal agents. The median/mode MIC for itraconazole and isavuconazole were ≥16 mg/l for all three pathogens. Based on limited data, these fungi are emerging. Invasive fusariosis increased from 0.08 cases/100 000 admissions to 0.22 cases/100 000 admissions over the time periods of 2000-2009 and 2010-2015, respectively, and in lung transplant recipients, Scedosporium spp. and L. prolificans were only detected from 2014 onwards. Global surveillance to better delineate antifungal susceptibility, risk factors, sequelae, and outcomes is required.

Exploring synergy between azole antifungal drugs and statins for Candida auris.

BACKGROUND: Global emergence of rapidly developing resistance to multiple antifungal drugs and high mortality pose challenges to the treatment of invasive Candida auris infections. New therapeutic approaches are needed, such as repurposing drugs including combination with antifungals. Statins have been reported to exert antifungal effects against various Candida species. OBJECTIVES: Our study investigated potential synergy between the statins (rosuvastatin and fluvastatin) and azoles (voriconazole, posaconazole and isavuconazole) on clinical isolates of C. auris. METHODS: Twenty-one clinical isolates of C. auris were obtained. Chequerboard assays based on the CLSI broth microdilution method were used to assess synergy based on FIC index (FICI) calculations of MICs of individual drugs and in combinations. RESULTS: Single drug geometric mean (GM) MICs of fluvastatin and rosuvastatin were ≥128 mg/L in all 21 isolates. GM (range) MICs of posaconazole, voriconazole and isavuconazole were 0.259 (0.016-1 mg/L), 0.469 (0.016-2 mg/L) and 0.085 (0.004-1 mg/L), respectively. Combination of azoles with fluvastatin showed synergy in 70%-90% of C. auris isolates. In particular, voriconazole/fluvastatin resulted in 16-fold reduction in voriconazole MIC and synergy in 14/21 (67%) isolates. Posaconazole/fluvastatin resulted in 8-fold reduction in posaconazole MIC and synergy in 19/21 (90%) isolates.Combining rosuvastatin with the azoles also showed synergy against C. auris in 40%-60% of the isolates and additive effect in 40%-50%. None of the combinations was antagonistic. CONCLUSIONS: Our results provide a rationale for pursuing in vivo synergy tests as well as clinical studies to explore tolerability, treatment outcomes, optimal dose and exposure targets.

Introducing 1,3-beta-d-glucan for screening and diagnosis of invasive fungal diseases in Australian high-risk haematology patients: is there a clinical benefit?

BACKGROUND: Early, accurate diagnosis of invasive fungal disease (IFD) improves clinical outcomes. 1,3-beta-d-glucan (BDG) (Fungitell, Associates of Cape Cod, Inc., Falmouth, MA, USA) detection can improve IFD diagnosis but has been unavailable in Australia. AIMS: To assess performance of serum BDG for IFD diagnosis in a high-risk Australian haematology population. METHODS: We compared the diagnostic value of weekly screening of serum BDG with screening by Aspergillus polymerase chain reaction and Aspergillus galactomannan in 57 at-risk episodes for the diagnosis of IFD (proven, probable, possible IFD). RESULTS: IFD episodes were: proven (n = 4); probable (n = 4); possible (n = 18); and no IFD (n = 31). Using two consecutive BDG results of ≥80 pg/mL to call a result 'positive', the sensitivity, specificity, positive predictive value and negative predictive value was 37.5%, 64.5%, 23.1% and 80.7% respectively. For invasive aspergillosis, test performance increased to 50%, 90.3%, 57.1% and 87.5% respectively if any two of serum BDG/Aspergillus polymerase chain reaction/galactomannan yielded a 'positive' result. In proven/probable IFD, five of eight episodes returned a positive BDG result earlier (mean 6.6 days) than other diagnostic tests. False-negative BDG results occurred in three of eight episodes of proven/probable IFD, and false positive in 10 of 31 patients with no IFD. Erratic patterns of BDG values predicted false positive results (P = 0.03). Using serum BDG results, possible IFD were reassigned to either 'no' or 'probable' IFD in 44% cases. Empiric anti-fungal therapy use may have been optimised by BDG monitoring in 38.5% of courses. CONCLUSIONS: The BDG assay can add diagnostic speed and value but was hampered by low sensitivity and positive predictive value in Australian haematology patients.

Azole-resistant Aspergillus fumigatus is highly prevalent in the environment of Vietnam, with marked variability by land use type.

Azole-resistant environmental Aspergillus fumigatus presents a threat to public health but the extent of this threat in Southeast Asia is poorly described. We conducted environmental surveillance in the Mekong Delta region of Vietnam, collecting air and ground samples across key land-use types, and determined antifungal susceptibilities of Aspergillus section Fumigati (ASF) isolates and azole concentrations in soils. Of 119 ASF isolates, 55% were resistant (or non-wild type) to itraconazole, 65% to posaconazole and 50% to voriconazole. Azole resistance was more frequent in A. fumigatus sensu stricto isolates (95%) than other ASF species (32%). Resistant isolates and agricultural azole residues were overrepresented in samples from cultivated land. cyp51A gene sequence analysis showed 38/56 resistant A. fumigatus sensu stricto isolates carried known resistance mutations, with TR34 /L98H most frequent (34/38).

Consensus guidelines for the diagnosis and management of invasive aspergillosis, 2021.

Invasive aspergillosis (IA) in haematology/oncology patients presents as primary infection or breakthrough infection, which can become refractory to antifungal treatment and has a high associated mortality. Other emerging patient risk groups include patients in the intensive care setting with severe respiratory viral infections, including COVID-19. These guidelines present key diagnostic and treatment recommendations in light of advances in knowledge since the previous guidelines in 2014. Culture and histological-based methods remain central to the diagnosis of IA. There is increasing evidence for the utility of non-culture methods employing fungal biomarkers in pre-emptive screening for infection, as well as for IA diagnosis when used in combination. Although azole resistance appears to be uncommon in Australia, susceptibility testing of clinical Aspergillus fumigatus complex isolates is recommended. Voriconazole remains the preferred first-line antifungal agent for treating primary IA, including for extrapulmonary disease. Recommendations for paediatric treatment broadly follow those for adults. For breakthrough and refractory IA, a change in class of antifungal agent is strongly recommended, and agents under clinical trial may need to be considered. Newer immunological-based imaging modalities warrant further study, while surveillance for IA and antifungal resistance remain essential to informing the relevance of current treatment recommendations.

Risk factors for candidaemia: A prospective multi-centre case-control study.

OBJECTIVES: Candidaemia carries a mortality of up to 40% and may be related to increasing complexity of medical care. Here, we determined risk factors for the development of candidaemia. METHODS: We conducted a prospective, multi-centre, case-control study over 12 months. Cases were aged ≥18 years with at least one blood culture positive for Candida spp. Each case was matched with two controls, by age within 10 years, admission within 6 months, admitting unit, and admission duration at least as long as the time between admission and onset of candidaemia. RESULTS: A total of 118 incident cases and 236 matched controls were compared. By multivariate analysis, risk factors for candidaemia included neutropenia, solid organ transplant, significant liver, respiratory or cardiovascular disease, recent gastrointestinal, biliary or urological surgery, central venous access device, intravenous drug use, urinary catheter and carbapenem receipt. CONCLUSIONS: Risk factors for candidaemia derive from the infection source, carbapenem use, host immune function and organ-based co-morbidities. Preventive strategies should target iatrogenic disruption of mucocutaneous barriers and intravenous drug use.

Comparing immunochromatography with latex antigen agglutination testing for the diagnosis of cryptococcosis in cats, dogs and koalas.

Although the point-of-care cryptococcal antigen lateral flow assay (LFA) has revolutionized the diagnosis of cryptococcosis in human patients, to date there has been no large-scale examination of this test in animals. We therefore assessed the LFA in cats, dogs and koalas suspected of having cryptococcosis. In sum, 528 serum specimens (129 from cats, 108 from dogs, 291 from koalas) were tested using the LFA and one of two commercially available latex cryptococcal antigen agglutination test (LCAT) kits. The LCAT is a proven and well-accepted method in veterinary patients and therefore taken as the "gold standard" against which the LFA was compared. The LFA achieved a sensitivity of 92%, 100%, and 98% in cats, dogs, and koalas, respectively, with corresponding negative predictive values of 94%, 100%, and 98%. The specificity of the LFA was 81%, 84%, and 62% for cats, dogs, and koalas, respectively, with corresponding positive predictive values of 76%, 48%, and 69%. These findings suggest the most appropriate role for the LFA is as a screening test to rule out a diagnosis of cryptococcosis in cats, dogs, and koalas. Point-of-care accessibility makes it equally suited for use in the field and as a cage-side test in veterinary hospitals. The suboptimal specificity of the LFA makes it less suited to definitive confirmation of cryptococcosis in animals; therefore, all LFA-positive test results should be confirmed by LCAT testing. The discrepancy between these observations and the high specificity of the LFA in humans may reflect differences in the host-pathogen interactions amongst the species.

Long-read sequencing based clinical metagenomics for the detection and confirmation of Pneumocystis jirovecii directly from clinical specimens: A paradigm shift in mycological diagnostics.

The advent of next generation sequencing technologies has enabled the characterization of the genetic content of entire communities of organisms, including those in clinical specimens, without prior culturing. The MinION from Oxford Nanopore Technologies offers real-time, direct sequencing of long DNA fragments directly from clinical samples. The aim of this study was to assess the ability of unbiased, genome-wide, long-read, shotgun sequencing using MinION to identify Pneumocystis jirovecii directly from respiratory tract specimens and to characterize the associated mycobiome. Pneumocystis pneumonia (PCP) is a life-threatening fungal disease caused by P. jirovecii. Currently, the diagnosis of PCP relies on direct microscopic or real-time quantitative polymerase chain reaction (PCR) examination of respiratory tract specimens, as P. jirovecii cannot be cultured readily in vitro. P. jirovecii DNA was detected in bronchoalveolar lavage (BAL) and induced sputum (IS) samples from three patients with confirmed PCP. Other fungi present in the associated mycobiome included known human pathogens (Aspergillus, Cryptococcus, Pichia) as well as commensal species (Candida, Malassezia, Bipolaris). We have established optimized sample preparation conditions for the generation of high-quality data, curated databases, and data analysis tools, which are key to the application of long-read MinION sequencing leading to a fundamental new approach in fungal diagnostics.

The Fungal PCR Initiative's evaluation of in-house and commercial Pneumocystis jirovecii qPCR assays: Toward a standard for a diagnostics assay.

Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal PCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicenter and monocenter evaluation of PCP qPCR assays was performed. For the multicenter study, 16 reference laboratories from eight different countries, performing 20 assays analyzed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads, respectively). The monocenter study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, reverse transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation.

Diagnosis, management and prevention of Candida auris in hospitals: position statement of the Australasian Society for Infectious Diseases.

Candida auris is an emerging drug-resistant yeast responsible for hospital outbreaks. This statement reviews the evidence regarding diagnosis, treatment and prevention of this organism and provides consensus recommendations for clinicians and microbiologists in Australia and New Zealand. C. auris has been isolated in over 30 countries (including Australia). Bloodstream infections are the most frequently reported infections. Infections have crude mortality of 30-60%. Acquisition is generally healthcare-associated and risks include underlying chronic disease, immunocompromise and presence of indwelling medical devices. C. auris may be misidentified by conventional phenotypic methods. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry or sequencing of the internal transcribed spacer regions and/or the D1/D2 regions of the 28S ribosomal DNA are therefore required for definitive laboratory identification. Antifungal drug resistance, particularly to fluconazole, is common, with variable resistance to amphotericin B and echinocandins. Echinocandins are currently recommended as first-line therapy for infection in adults and children ≥2 months of age. For neonates and infants <2 months of age, amphotericin B deoxycholate is recommended. Healthcare facilities with C. auris should implement a multimodal control response. Colonised or infected patients should be isolated in single rooms with Standard and Contact Precautions. Close contacts, patients transferred from facilities with endemic C. auris or admitted following stay in overseas healthcare institutions should be pre-emptively isolated and screened for colonisation. Composite swabs of the axilla and groin should be collected. Routine screening of healthcare workers and the environment is not recommended. Detergents and sporicidal disinfectants should be used for environmental decontamination.

Surveillance for azole resistance in clinical and environmental isolates of Aspergillus fumigatus in Australia and cyp51A homology modelling of azole-resistant isolates.

Background: The prevalence of azole resistance in Aspergillus fumigatus is uncertain in Australia. Azole exposure may select for resistance. We investigated the frequency of azole resistance in a large number of clinical and environmental isolates. Methods: A. fumigatus isolates [148 human, 21 animal and 185 environmental strains from air (n = 6) and azole-exposed (n = 64) or azole-naive (n = 115) environments] were screened for azole resistance using the VIPcheck™ system. MICs were determined using the Sensititre™ YeastOne YO10 assay. Sequencing of the Aspergillus cyp51A gene and promoter region was performed for azole-resistant isolates, and cyp51A homology protein modelling undertaken. Results: Non-WT MICs/MICs at the epidemiological cut-off value of one or more azoles were observed for 3/148 (2%) human isolates but not amongst animal, or environmental, isolates. All three isolates grew on at least one azole-supplemented well based on VIPcheck™ screening. For isolates 9 and 32, the itraconazole and posaconazole MICs were 1 mg/L (voriconazole MICs 0.12 mg/L); isolate 129 had itraconazole, posaconazole and voriconazole MICs of >16, 1 and 8 mg/L, respectively. Soil isolates from azole-exposed and azole-naive environments had similar geometric mean MICs of itraconazole, posaconazole and voriconazole (P > 0.05). A G54R mutation was identified in the isolates exhibiting itraconazole and posaconazole resistance, and the TR34/L98H mutation in the pan-azole-resistant isolate. cyp51A modelling predicted that the G54R mutation would prevent binding of itraconazole and posaconazole to the haem complex. Conclusions: Azole resistance is uncommon in Australian clinical and environmental A. fumigatus isolates; further surveillance is indicated.

In vitro activity of the novel antifungal compound F901318 against Australian Scedosporium and Lomentospora fungi.

We determined the in vitro activity of the novel orotomide antifungal, F901318, against 30 Lomentospora prolificans, 20 Scedosporium apiospermum, 7 S. aurantiacum, and 3 S. boydii, isolates in comparison with standard antifungals. Against L. prolificans, F901318 was the most potent compound (MIC90 0.25 µg/ml); the geometric mean MIC (0.26 µg/ml) was significantly lower (23-80-fold) than those of itraconazole, voriconazole, posaconazole, and isavuconazole (all P < .001), and amphotericin B (P < .05). F901318 also had good activity against S. apiospermum, S. aurantiacum, and S. boydii, comparable to that of voriconazole and posaconazole but was more active than isavuconazole for all three species.

Online Databases for Taxonomy and Identification of Pathogenic Fungi and Proposal for a Cloud-Based Dynamic Data Network Platform.

The increase in public online databases dedicated to fungal identification is noteworthy. This can be attributed to improved access to molecular approaches to characterize fungi, as well as to delineate species within specific fungal groups in the last 2 decades, leading to an ever-increasing complexity of taxonomic assortments and nomenclatural reassignments. Thus, well-curated fungal databases with substantial accurate sequence data play a pivotal role for further research and diagnostics in the field of mycology. This minireview aims to provide an overview of currently available online databases for the taxonomy and identification of human and animal-pathogenic fungi and calls for the establishment of a cloud-based dynamic data network platform.

PCR-based tests for the early diagnosis of sepsis. Where do we stand?

PURPOSE OF REVIEW: Bloodstream infections are a major cause of hospital and ICU admission with high morbidity and mortality; however, early and targeted antimicrobial therapy reduces mortality in high-risk patients. This article focuses on the diagnosis of bloodstream infections by PCR-based approaches at an early stage to enable prompt treatment and prevent organ dysfunction. RECENT FINDINGS: PCR systems offering highly multiplexed targeting of bacterial and/or fungal pathogens (in whole blood) offer the best opportunity for clinical impact, as informed decisions can be made within 4-8 h of the blood draw. Although more rapid, these systems are typically associated with lower sensitivity and specificity than postculture detection methods which rely on microbial growth. Additionally, unlike postculture methods, detection directly from blood is not prone to misleading results because of concurrent (or previous) therapy, which limit clinical relevance. SUMMARY: Rapid and accurate identification of the cause of sepsis is essential in improving patient outcomes. Early identification of these pathogens by nucleic acid detection assays directly from blood samples remains key to achieving this, particularly if taken at the time of presentation. Selection of the most suitable PCR system is typically influenced by local epidemiology and by the resources of the testing laboratory.

Changing epidemiology of candidaemia in Australia.

Objectives: Knowledge of contemporary epidemiology of candidaemia is essential. We aimed to identify changes since 2004 in incidence, species epidemiology and antifungal susceptibilities of Candida spp. causing candidaemia in Australia. Methods: These data were collected from nationwide active laboratory-based surveillance for candidaemia over 1 year (within 2014-2015). Isolate identification was by MALDI-TOF MS supplemented by DNA sequencing. Antifungal susceptibility testing was performed using Sensititre YeastOne™. Results: A total of 527 candidaemia episodes (yielding 548 isolates) were evaluable. The mean annual incidence was 2.41/105 population. The median patient age was 63 years (56% of cases occurred in males). Of 498 isolates with confirmed species identity, Candida albicans was the most common (44.4%) followed by Candida glabrata complex (26.7%) and Candida parapsilosis complex (16.5%). Uncommon Candida species comprised 25 (5%) isolates. Overall, C. albicans (>99%) and C. parapsilosis (98.8%) were fluconazole susceptible. However, 16.7% (4 of 24) of Candida tropicalis were fluconazole- and voriconazole-resistant and were non-WT to posaconazole. Of C. glabrata isolates, 6.8% were resistant/non-WT to azoles; only one isolate was classed as resistant to caspofungin (MIC of 0.5 mg/L) by CLSI criteria, but was micafungin and anidulafungin susceptible. There was no azole/echinocandin co-resistance. Conclusions: We report an almost 1.7-fold proportional increase in C. glabrata candidaemia (26.7% versus 16% in 2004) in Australia. Antifungal resistance was generally uncommon, but azole resistance (16.7% of isolates) amongst C. tropicalis may be emerging.

Molecular diagnosis of Pneumocystis pneumonia in dogs.

Pneumocystis pneumonia (PCP) is a life-threatening fungal disease that can occur in dogs. The aim of this study was to provide a preliminary genetic characterisation of Pneumocystis carinii f.sp.'canis' (P. canis) in dogs and thereby develop a reliable molecular protocol to definitively diagnose canine PCP. We investigated P. canis in a variety of lung specimens from dogs with confirmed or strongly suspected PCP (Group 1, n = 16), dogs with non-PCP lower respiratory tract problems (Group 2, n = 65) and dogs not suspected of having PCP or other lower respiratory diseases (Group 3, n = 11). Presence of Pneumocystis DNA was determined by nested PCR of the large and small mitochondrial subunit rRNA loci and by a real-time quantitative polymerase chain reaction (qPCR) assay developed using a new set of primers. Molecular results were correlated with the presence of Pneumocystis morphotypes detected in cytological/histological preparations. Pneumocystis DNA was amplified from 13/16 PCP-suspected dogs (Group 1) and from 4/76 dogs of control Groups 2 and 3 (combined). The latter four dogs were thought to have been colonized by P. canis. Comparison of CT values in 'infected' versus 'colonized' dogs was consistent with this notion, with a distinct difference in molecular burden between groups (CT ≤ 26 versus CT range (26