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.

Multi-locus sequencing typing reveals geographically related intraspecies variability of Sporothrix brasiliensis.

Sporotrichosis is a subcutaneous mycosis caused by pathogenic Sporothrix species. Among them, Sporothrix brasiliensis is the main species associated with endemic regions in South America, especially Brazil. It is highly virulent and can be spread through zoonotic transmission. Molecular epidemiological surveys are needed to determine the extent of genetic variation, to investigate outbreaks, and to identify genotypes associated with antifungal resistance and susceptibility. This study investigated the sequence variation of different constitutive genes and established a novel multilocus sequence typing (MLST) scheme for S. brasiliensis. Specific primers were designed for 16 genes using Primer-BLAST software based on the genome sequences of three S. brasiliensis strains (ATCC MYA-4823, A001 and A005). Ninety-one human, animal, and environmental S. brasiliensis isolates from different Brazilian geographic regions (South, Southeast, Midwest and Northeast) andtwo isolates from Paraguay were sequenced. The loci that presented the highest nucleotide diversity (π) were selected for the MLST scheme. Among the 16 studied genetic loci, four presented increased π value and were able to distinguish all S. brasiliensis isolates into seven distinct haplotypes. The PCR conditions were standardized for four loci. Some of the obtained haplotypes were associated with the geographic origin of the strains. This study presents an important advance in the understanding of this important agent of sporotrichosis in Brazil. It significantly increased the discriminatory power for genotyping of S. brasiliensis isolates, and enabled new contributions to the epidemiological studies of this human and animal pathogen in Brazil and in other countries.

A conceptual framework for nomenclatural stability and validity of medically important fungi: a proposed global consensus guideline for fungal name changes supported by ABP, ASM, CLSI, ECMM, ESCMID-EFISG, EUCAST-AFST, FDLC, IDSA, ISHAM, MMSA, and MSGERC.

The rapid pace of name changes of medically important fungi is creating challenges for clinical laboratories and clinicians involved in patient care. We describe two sources of name change which have different drivers, at the species versus the genus level. Some suggestions are made here to reduce the number of name changes. We urge taxonomists to provide diagnostic markers of taxonomic novelties. Given the instability of phylogenetic trees due to variable taxon sampling, we advocate to maintain genera at the largest possible size. Reporting of identified species in complexes or series should where possible comprise both the name of the overarching species and that of the molecular sibling, often cryptic species. Because the use of different names for the same species will be unavoidable for many years to come, an open access online database of the names of all medically important fungi, with proper nomenclatural designation and synonymy, is essential. We further recommend that while taxonomic discovery continues, the adaptation of new name changes by clinical laboratories and clinicians be reviewed routinely by a standing committee for validation and stability over time, with reference to an open access database, wherein reasons for changes are listed in a transparent way.

Molecular Detection of Histoplasma capsulatum in Antarctica.

We detected Histoplasma capsulatum in soil and penguin excreta in the Antarctic Peninsula by sequencing after performing species-specific PCR, confirming previous observations that this pathogen occurs more broadly than suspected. This finding highlights the need for surveillance of emerging agents of systemic mycoses and their transmission among regions, animals, and humans in Antarctica.

In depth search of the Sequence Read Archive database reveals global distribution of the emerging pathogenic fungus Scedosporium aurantiacum.

Scedosporium species are emerging opportunistic fungal pathogens causing various infections mainly in immunocompromised patients, but also in immunocompetent individuals, following traumatic injuries. Clinical manifestations range from local infections, such as subcutaneous mycetoma or bone and joint infections, to pulmonary colonization and severe disseminated diseases. They are commonly found in soil and other environmental sources. To date S. aurantiacum has been reported only from a handful of countries. To identify the worldwide distribution of this species we screened publicly available sequencing data from fungal metabarcoding studies in the Sequence Read Archive (SRA) of The National Centre for Biotechnology Information (NCBI) by multiple BLAST searches. S. aurantiacum was found in 26 countries and two islands, throughout every climatic region. This distribution is like that of other Scedosporium species. Several new environmental sources of S. aurantiacum including human and bovine milk, chicken and canine gut, freshwater, and feces of the giant white-tailed rat (Uromys caudimaculatus) were identified. This study demonstrated that raw sequence data stored in the SRA database can be repurposed using a big data analysis approach to answer biological questions of interest. LAY SUMMARY: To understand the distribution and natural habitat of S. aurantiacum, species-specific DNA sequences were searched in the SRA database. Our large-scale data analysis illustrates that S. aurantiacum is more widely distributed than previously thought and new environmental sources were identified.

Host transmission dynamics of first- and third-stage Angiostrongylus cantonensis larvae in Bullastra lessoni.

Given the importance of angiostrongyliasis as an emerging infectious disease of humans, companion animals, and wildlife, the current study focused on the transmission dynamics of first- and third-stage larvae of the parasitic nematode, Angiostrongylus cantonensis. The migration of infective larvae and their subsequent distribution within the Lymnaeidae snail, Bullastra lessoni, were investigated over time using microscopic examination of histological sections and fresh tissue. Snails were divided into four anatomical regions: (i) anterior and (ii) posterior cephalopedal masses, (iii) mantle skirt and (iv) visceral mass. The viability of free-swimming third-stage larvae, after their release from snail tissues, was evaluated in vitro by propidium iodide staining and infectivity by in vivo infection of Wistar rats. Snails were sequentially dissected over time to assess the number and anatomical distribution of larvae within each snail and hence infer their migration pathway. Herein, ongoing larval migratory activity was detected over 28 days post-infection. A comparison of infection rates and the larval distribution within the four designated snail regions demonstrated a significant relationship between anatomical region and density of infective larvae, with larvae mostly distributed in the anterior cephalopedal mass (43.6 ± 10.8%) and the mantle skirt (33.0 ± 8.8%). Propidium iodide staining showed that free-swimming third-stage larvae retained viability for between 4 and 8 weeks when stored under laboratory conditions. In contrast to viability, larval infectivity in rats remained for up to 2 weeks only. Knowledge gained from the current work could provide information on the development of new approaches to controlling the transmission of this parasite.

Plasmodium falciparum Histidine-Rich Protein 2 and 3 Gene Deletions in Strains from Nigeria, Sudan, and South Sudan.

Deletion of histidine-rich protein genes pfhrp2/3 in Plasmodium falciparum causes infections to go undetected by HRP2-based malaria rapid diagnostic tests. We analyzed P. falciparum malaria cases imported to Australia (n = 210, collected 2010-2018) for their pfhrp2/3 status. We detected gene deletions in patients from 12 of 25 countries. We found >10% pfhrp2-deletion levels in those from Nigeria (13.3%, n = 30), Sudan (11.2%, n = 39), and South Sudan (17.7%, n = 17) and low levels of pfhrp3 deletion from Sudan (3.6%) and South Sudan (5.9%). No parasites with pfhrp2/3 double deletions were detected. Microsatellite typing of parasites from Nigeria, Sudan, and South Sudan revealed low relatedness among gene-deleted parasites, indicating independent emergences. The gene deletion proportions signify a risk of false-negative HRP2-RDT results. This study's findings warrant surveillance to determine whether the prevalence of gene-deleted parasites justifies switching malaria rapid diagnostic tests in Nigeria, Sudan, and South Sudan.

Pneumocystis jirovecii genetic diversity in a Spanish tertiary hospital.

Pneumocystis jirovecii is associated with non-noxious colonization or severe pneumonia in immunocompromised hosts. Epidemiological investigations have been hampered by the lack of a standardized typing scheme. Thus, only partial molecular data on Spanish P. jirovecii cases are available. Recently, a new ISHAM consensus multilocus sequence typing scheme (MLST) targeting ß-TUB, mt26S, CYB, and SOD with a publicly accessible database has been launched to overcome this problem. The molecular epidemiology of P. jirovecii from immunocompromised patients either colonized (n = 50) or having pneumonia (n = 36) seen between 2014 and 2018 at a single center in Barcelona, Spain, was studied. The new ISHAM consensus MSLT scheme was used to investigate the local epidemiology and identify possible unnoticed outbreaks. Mutations in the DHPS gene, not included in the scheme but giving information about potential sulfa treatment failure, were also studied. The study assigned 32 sequence types (ST) to 72.2% pneumonia and 56% colonization cases. The most frequent STs were ST21 (18.5%), ST22 (14.8%), and ST37(14.8%). For non-unique STs, ST3, ST30 and ST31 were found only in pneumonia cases, whereas ST27 was associated exclusively to colonizations. Despite 38 patients sharing similar STs, only two were involved in a potential cross transmission event. No DHPS mutations were identified. The new consensus typing scheme was useful to ascertain the molecular epidemiology of P. jirovecii in our center revealing a high genetic diversity and the potential association of specific STs to colonization and pneumonia cases. LAY SUMMARY: A newly described MLST scheme aims at providing a standardized tool to study and compare Pneumocystis jirovecii epidemiology. A high diversity among P. jirovecii isolates from patients in Barcelona, Spain, and a potential association between specific STs and infection/colonization were identified.

Consensus guidelines for the diagnosis and management of invasive fungal disease due to moulds other than Aspergillus in the haematology/oncology setting, 2021.

Invasive fungal disease (IFD) due to moulds other than Aspergillus is a significant cause of mortality in patients with malignancies or post haemopoietic stem cell transplantation. The current guidelines focus on the diagnosis and management of the common non-Aspergillus moulds (NAM), such as Mucorales, Scedosporium species (spp.), Lomentospora prolificans and Fusarium spp. Rare but emerging NAM including Paecilomyces variotii, Purpureocillium lilacinum and Scopulariopsis spp. are also reviewed. Culture and histological examination of tissue biopsy specimens remain the mainstay of diagnosis, but molecular methods are increasingly being used. As NAM frequently disseminate, blood cultures and skin examination with biopsy of any suspicious lesions are critically important. Treatment requires a multidisciplinary approach with surgical debridement as a central component. Other management strategies include control of the underlying disease/predisposing factors, augmentation of the host response and the reduction of immunosuppression. Carefully selected antifungal therapy, guided by susceptibility testing, is critical to cure. We also outline novel antifungal agents still in clinical trial which offer substantial potential for improved outcomes in the future. Paediatric recommendations follow those of adults. Ongoing epidemiological research, improvement in diagnostics and the development of new antifungal agents will continue to improve the poor outcomes that have been traditionally associated with IFD due to NAM.

New multilocus sequence typing primers to enable genotyping of AD hybrids within the Cryptococcus neoformans species complex.

Although AD hybrids within the Cryptococcus neoformans species complex represent about 20% of the isolates identified in Europe, phylogenetic and population genetic studies are lacking due to the inability to use the standardized typing method. The aim of the present study was to design new molecular type specific primers in order to apply the standard ISHAM consensus multilocus sequence typing (MLST) scheme to AD hybrids. The new primers are able to specifically amplify VNI and VNIV alleles of the seven MLST loci in both haploid and diploid or aneuploid hybrid strains. This study forms the basis for future molecular epidemiology studies of AD hybrids. LAY ABSTRACT: We designed and tested new specific primers to amplify the two alleles of each of the seven MLST loci in C. neoformans species complex hybrids. The sequences obtained from hybrids can be compared with those present in the Cryptococcus global MLST database for future molecular epidemiology studies.

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.

Genotypes and population genetics of cryptococcus neoformans and cryptococcus gattii species complexes in Europe and the mediterranean area.

A total of 476 European isolates (310 Cryptococcus neoformans var. grubii, 150 C. neoformans var. neoformans, and 16 C. gattii species complex) from both clinical and environmental sources were analyzed by multi-locus sequence typing. Phylogenetic and population genetic analyses were performed. Sequence analysis identified 74 sequence types among C. neoformans var. neoformans (VNIV), 65 among C. neoformans var. grubii (56 VNI, 8 VNII, 1 VNB), and 5 among the C. gattii species complex (4 VGI and 1 VGIV) isolates. ST23 was the most frequent genotype (22%) among VNI isolates which were mostly grouped in a large clonal cluster including 50% of isolates. Among VNIV isolates, a predominant genotype was not identified. A high percentage of autochthonous STs were identified in both VNI (71%) and VNIV (96%) group of isolates. The 16 European C. gattii species complex isolates analyzed in the present study originated all from the environment and all belonged to a large cluster endemic in the Mediterranean area. Population genetic analysis confirmed that VNI group of isolates were characterized by low variability and clonal expansion while VNIV by a higher variability and a number of recombination events. However, when VNI and VNIV environmental isolates were compared, they showed a similar population structure with a high percentage of shared mutations and the absence of fixed mutations. Also linkage disequilibrium analysis reveals differences between clinical and environmental isolates showing a key role of PLB1 allele combinations in host infection as well as the key role of LAC1 allele combinations for survival of the fungus in the environment. The present study shows that genetic comparison of clinical and environmental isolates represents a first step to understand the genetic characteristics that cause the shift of some genotypes from a saprophytic to a parasitic life style.

Molecular detection of drug resistant malaria in Southern Thailand.

BACKGROUND: Drug resistance within the major malaria parasites Plasmodium vivax and Plasmodium falciparum threatens malaria control and elimination in Southeast Asia. Plasmodium vivax first-line treatment drug is chloroquine together with primaquine, and the first-line treatment for P. falciparum malaria is artemisinin in combination with a partner drug. Plasmodium vivax and P. falciparum parasites resistant to their respective first-line therapies are now found within Southeast Asia. The resistance perimeters may include high transmission regions of Southern Thailand which are underrepresented in surveillance efforts. METHODS: This study investigated blood samples from malaria centres in Southern Thailand. Genetic loci associated with drug resistance were amplified and sequenced. Drug resistance associated genes Pvmdr1, Pvcrt-o, Pvdhfr, and Pvdhps were characterized for 145 cases of P. vivax malaria, as well as the artemisinin resistance-associated Pfkelch13 gene from 91 cases of P. falciparum malaria. RESULTS: Plasmodium vivax samples from Southern Thai provinces showed numerous chloroquine and antifolate resistance-associated mutations, including SNP and Pvcrt-o K10-insertion combinations suggestive of chloroquine resistant P. vivax phenotypes. A high proportion of the C580Y coding mutation (conferring artemisinin resistance) was detected in P. falciparum samples originating from Ranong and Yala (where the mutation was previously unreported). CONCLUSIONS: The results demonstrate a risk of chloroquine and antifolate resistant P. vivax phenotypes in Southern Thailand, and artemisinin resistant P. falciparum observed as far south as the Thai-Malaysian border region. Ongoing surveillance of antimalarial drug resistance markers is called for in Southern Thailand to inform case management.

Database establishment for the secondary fungal DNA barcode translational elongation factor 1α (TEF1α) 1.

With new or emerging fungal infections, human and animal fungal pathogens are a growing threat worldwide. Current diagnostic tools are slow, non-specific at the species and subspecies levels, and require specific morphological expertise to accurately identify pathogens from pure cultures. DNA barcodes are easily amplified, universal, short species-specific DNA sequences, which enable rapid identification by comparison with a well-curated reference sequence collection. The primary fungal DNA barcode, ITS region, was introduced in 2012 and is now routinely used in diagnostic laboratories. However, the ITS region only accurately identifies around 75% of all medically relevant fungal species, which has prompted the development of a secondary barcode to increase the resolution power and suitability of DNA barcoding for fungal disease diagnostics. The translational elongation factor 1α (TEF1α) was selected in 2015 as a secondary fungal DNA barcode, but it has not been implemented into practice, due to the absence of a reference database. Here, we have established a quality-controlled reference database for the secondary barcode that together with the ISHAM-ITS database, forms the ISHAM barcode database, available online at http://its.mycologylab.org/ . We encourage the mycology community for active contributions.

Multi-locus sequence typing as a tool to investigate environmental sources of infection for cryptococcosis in captive birds.

A systematic investigation into environmental sources of infection was conducted at an Australian zoological park after cryptococcosis, caused by Cryptococcus gattii VGI, was diagnosed in a red-tailed black cockatoo (Calyptorhynchus banksii) residing in a large aviary with a diverse range of other avian species. A single tree with an extensive hollow was identified as the likely source of infection based on heavy culture of C. gattii VGI, multi-locus sequence typing and phylogenetic analysis of environmental and disease-related isolates. This led to the careful removal of the tree to reduce the risk of future cases of cryptococcosis in this aviary.

Molecular detection of Pneumocystis in the lungs of cats.

The genus Pneumocystis comprises potential pathogens that reside normally in the lungs of a wide range of mammals. Although they generally behave as transient or permanent commensals, they can occasionally cause life-threatening pneumonia (Pneumocystis pneumonia; PCP) in immunosuppressed individuals. Several decades ago, the presence of Pneumocystis morphotypes (trophic forms and cysts) was described in the lungs of normal cats and cats with experimentally induced symptomatic PCP (after immunosuppression by corticosteroids); yet to date spontaneous or drug-induced PCP has not been described in the clinical feline literature, despite immunosuppression of cats by long-standing retrovirus infections or after kidney transplantation. In this study, we describe the presence of Pneumocystis DNA in the lungs of normal cats (that died of various unrelated causes; n = 84) using polymerase chain reactions (PCRs) targeting the mitochondrial small and large subunit ribosomal RNA gene (mtSSU rRNA and mtLSU rRNA). The presence of Pneumocystis DNA was confirmed by sequencing in 24/84 (29%) cats, with evidence of two different sequence types (or lineages). Phylogenetically, lineage1 (L1; 19 cats) and lineage 2 (L2; 5 cats) formed separate clades, clustering with Pneumocystis from domestic pigs (L1) and carnivores (L2), respectively. Results of the present study support the notion that cats can be colonized or subclinically infected by Pneumocystis, without histological evidence of damage to the pulmonary parenchyma referable to pneumocystosis. Pneumocystis seems most likely an innocuous pathogen of cats' lungs, but its possible role in the exacerbation of chronic pulmonary disorders or viral/bacterial coinfections should be considered further in a clinical setting.

Prevalence of cryptococcal antigenemia and nasal colonization in a free-ranging koala population.

Cryptococcosis, caused by environmental fungi in the Cryptococcus neoformans and Cryptococcus gattii species complexes, affects a variety of hosts, including koalas (Phascolarctos cinereus). Cryptococcal antigenemia and nasal colonization are well characterized in captive koalas, but free-ranging populations have not been studied systematically. Free-ranging koalas (181) from the Liverpool Plains region of New South Wales, Australia, were tested for cryptococcal antigenemia (lateral flow immunoassay) and nasal colonization (bird seed agar culture). Results were related to environmental and individual koala characteristics. Eucalypt trees (14) were also randomly tested for the presence of Cryptococcus spp. by bird seed agar culture. In sum, 5.5% (10/181) and 6.6% (12/181) of koalas were positive for antigenemia and nasal colonization, respectively, on at least one occasion. And 64.3% (9/14) of eucalypts were culture-positive for Cryptococcus spp. URA5 restriction fragment length polymorphism analysis identified most isolates as C. gattii VGI, while C. neoformans VNI was only found in one koala and one tree. Colonized koalas were significantly more likely to test positive for antigenemia. No associations between antigenemia or colonization, and external environmental characteristics (the relative abundance of Eucalyptus camaldulensis and season), or individual koala characteristics (body condition, sex, and age), could be established, suggesting that antigenemia and colonization are random outcomes of host-pathogen-environment interactions. The relationship between positive antigenemia status and a relatively high abundance of E. camaldulensis requires further investigation. This study characterizes cryptococcosis in a free-ranging koala population, expands the ecological niche of the C. gattii/C. neoformans species complexes and highlights free-ranging koalas as important sentinels for this disease.

Effect of peptidases secreted by the opportunistic pathogen Scedosporium aurantiacum on human epithelial cells.

Peptidases secreted by a clinical high-virulence Scedosporium aurantiacum isolate (strain WM 06.482; CBS 136046) under normoxic and hypoxic conditions were separated via size-exclusion chromatography, and peptidase activities present in each fraction were determined using class-specific substrates. The fractions demonstrating peptidase activity were assessed for their effects on the attachment and viability of A549 human lung epithelial cells in vitro. Of the peptidases detected in the size-exclusion chromatography fractions, the elastase-like peptidase reduced cell viability, the chymotrypsin-like peptidase was associated with cell detachment, and the cysteine peptidases were able to abolish both cell attachment and viability. The loss of cell viability and attachment became more prominent with an increase in the peptidase activity and could also be specifically prevented by addition of class-specific peptidase inhibitors. Our findings indicate that peptidases secreted by S. aurantiacum can breach the human alveolar epithelial cell barrier and, thus, may have a role in the pathobiology of the organism.

Identification of 14-α-Lanosterol Demethylase (CYP51) in Scedosporium Species.

Scedosporium spp. cause infections (scedosporiosis) in both immunocompetent and immunocompromised individuals and may persistently colonize the respiratory tract in patients with cystic fibrosis (CF). They are less susceptible against azoles than are other molds, such as Aspergillus spp., suggesting the presence of resistance mechanisms. It can be hypothesized that the decreased susceptibility of Scedosporium spp. to azoles is also CYP51 dependent. Analysis of the Scedosporium apiospermum and Scedosporiumaurantiacum genomes revealed one CYP51 gene encoding the 14-α-lanosterol demethylase. This gene from 159 clinical or environmental Scedosporium isolates and three Lomentospora prolificans isolates has been sequenced and analyzed. The Scedosporium CYP51 protein clustered with the group of known CYP51B orthologues and showed species-specific polymorphisms. A tandem repeat in the 5' upstream region of Scedosporium CYP51 like that in Aspergillus fumigatus could not be detected. Species-specific amino acid alterations in CYP51 of Scedosporium boydii, Scedosporiumellipsoideum, Scedosporium dehoogii, and Scedosporiumminutisporum isolates were located at positions that have not been described as having an impact on azole susceptibility. In contrast, two of the three Sapiospermum-specific amino acid changes (Y136F and G464S) corresponded to respective mutations in A. fumigatus CYP51A at amino acid positions 121 and 448 (Y121F and G448S, respectively) that had been linked to azole resistance.