Search Results (66)

Cryptococcus is a genus of fungal pathogens that can infect and cause disease in a range of host species and is particularly prominent in koalas (Phascolarctos cinerus). Like other host species, koalas display a range of outcomes upon exposure to environmental Cryptococcus, from external nasal colonization to asymptomatic invasive infection and, in rare cases, severe clinical disease resulting in death. Host factors contributing to these varied outcomes are poorly understood. Due to their close relationship with eucalypt trees (a key environmental niche for Cryptococcus gattii) and suspected continual exposure to the pathogen, koalas provide a unique opportunity to examine host susceptibility in natural infections. Caspase recruitment domain-containing protein 9 (CARD9) is a key intracellular signaling protein in the fungal innate immune response. Humans with mutations in CARD9 succumb to several different severe and chronic fungal infections. This study is the first to sequence and explore CARD9 variation in multiple koalas using Sanger sequencing. Four CARD9 exons were successfully sequenced in 22 koalas from a New South Wales, Australia population. We found minimal variation between koalas across all four exons, an observation that was also made when CARD9 sequences were compared between koalas and six other species, including humans and mice. Ten single-nucleotide polymorphisms (SNP) were identified in this study and explored in the context of cryptococcal exposure outcomes. While we did not find any significant association with variation in cryptococcal outcomes, we found a high degree of conservation between species at several SNP loci that requires further investigation. The findings from this study lay the groundwork for further investigations of CARD9 and Cryptococcus both in koalas and other species, and highlight several considerations for future studies. Full article

The rapid and broad microbiological diagnosis of meningoencephalitis (ME) has been possible thanks to the development of multiplex PCR tests applied to cerebrospinal fluid (CSF). We aimed to assess a new multiplex PCR panel (the QIAstat-Dx ME panel), which we compared to conventional diagnostic tools and the Biofire FilmArray ME Panel. The pathogens analyzed using both methods were Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, Streptococcus pneumoniae, Enterovirus, herpes simplex virus 1–2, human herpesvirus 6, human parechovirus, varicella zoster virus, and Cryptococcus neoformans/gattii. We used sensitivity, specificity, PPV, NPV, and kappa correlation index parameters to achieve our objective. Fifty CSF samples from patients with suspected ME were included. When conventional methods were used, 28 CSF samples (56%) were positive. The sensitivity and specificity for QIAstat-Dx/ME were 96.43% (CI95%, 79.8–99.8) and 95.24% (75.2–99.7), respectively, whereas the PPV and NPV were 96.43% (79.8–99.8) and 95.24% (75.1–99.7), respectively. The kappa value was 91.67%. Conclusions: A high correlation of the QIAstat-Dx ME panel with reference methods was shown. QIAstat-Dx ME is a rapid-PCR technique to be applied in patients with suspected ME with a high accuracy. Full article

Infections caused by Cryptococcus neoformans and Cryptococcus gattii remain a challenge to our healthcare systems as they are still difficult to treat. In order to improve treatment success, in particular for infections that have disseminated to the central nervous system, a better understanding of the disease is needed, addressing questions like how it evolves from a pulmonary to a brain disease and how novel treatment approaches can be developed and validated. This requires not only clinical research and research on the microorganisms in a laboratory environment but also preclinical models in order to study cryptococci in the host. We provide an overview of available preclinical models, with particular emphasis on models of cryptococcosis in rodents. In order to further improve the characterization of rodent models, in particular the dynamic aspects of disease manifestation, development, and ultimate treatment, preclinical in vivo imaging methods are increasingly used, mainly in research for oncological, neurological, and cardiac diseases. In vivo imaging applications for fungal infections are rather sparse. A second aspect of this review is how research on models of cryptococcosis can benefit from in vivo imaging methods that not only provide information on morphology and tissue structure but also on function, metabolism, and cellular properties in a non-invasive way. Full article

Antifungal therapy, especially with the azoles, could promote the incidence of less susceptible isolates of Cryptococcus neoformans and C. gattii species complexes (SC), mostly in developing countries. Given that these species affect mostly the immunocompromised host, the infections are severe and difficult to treat. This review encompasses the following topics: 1. infecting species and their virulence, 2. treatment, 3. antifungal susceptibility methods and available categorical endpoints, 4. genetic mechanisms of resistance, 5. clinical resistance, 6. fluconazole minimal inhibitory concentrations (MICs), clinical outcome, 7. environmental influences, and 8. the relevance of host factors, including pharmacokinetic/pharmacodynamic (PK/PD) parameters, in predicting the clinical outcome to therapy. As of now, epidemiologic cutoff endpoints (ECVs/ECOFFs) are the most reliable antifungal resistance detectors for these species, as only one clinical breakpoint (amphotericin B and C. neoformans VNI) is available. Full article

Cryptococcosis in HIV-negative patients can be an opportunistic or endemic disease. There are no published studies on the use of the finger-prick whole blood (point-of-care) cryptococcal antigen lateral flow assay (CrAg LFA) for diagnosing cryptococcosis in HIV-negative patients. We conducted a case series study of HIV-negative patients with cryptococcosis in two centers in São Paulo, Brazil. The objectives were to identify the sensitivity of a finger-prick whole blood CrAg LFA and to describe the main characteristics of this population. We identified 30 HIV-negative patients with cryptococcosis [19 (63%), male; median age, 47 years]. Ten (33%) patients were immunosuppressed, ten (33%) had other comorbidities, and ten (33%) were apparently immunocompetent and without comorbidities. The distribution of the sites of cryptococcosis was as follows: the central nervous system, 90% (n = 27); pulmonary, 43% (n = 13); and other extrapulmonary sites, 40% (n = 12). The sensitivity of the finger-prick whole blood CrAg LFA for the diagnosis of cryptococcosis was 97% (29/30). Among 26 participants with cryptococcal meningitis, the sensitivity of testing cerebrospinal fluid was as follows: CrAg latex agglutination, 77% (20/26); CrAg LFA, 96% (25/26); and culture, 81% (21/26). Culture speciation identified Cryptococcus gattii in 16 (62%) cases, and all had a positive finger-prick whole blood CrAg LFA. This test presented high sensitivity to the diagnosis of cryptococcosis in HIV-negative patients, including those caused by C. gattii. Full article

Invasive fungal diseases are a public health problem. They affect a constantly increasing number of at-risk patients, and their incidence has risen in recent years. These opportunistic infections are mainly due to Candida sp. but less common or rare yeast infections should not be underestimated. These so-called “less common” yeasts include Ascomycota of the genera Candida (excluding the five major Candida species), Magnusiomyces/Saprochaete, Malassezia, and Saccharomyces, and Basidiomycota of the genera Cryptococcus (excluding the Cryptococcus neoformans/gattii complex members), Rhodotorula, and Trichosporon. The aim of this review is to (i) inventory the less common yeasts isolated in humans, (ii) provide details regarding the specific anatomical locations where they have been detected and the clinical characteristics of the resulting infections, and (iii) provide an update on yeast taxonomy. Of the total of 239,890 fungal taxa and their associated synonyms sourced from the MycoBank and NCBI Taxonomy databases, we successfully identified 192 yeasts, including 127 Ascomycota and 65 Basidiomycota. This repertoire allows us to highlight rare yeasts and their tropism for certain anatomical sites and will provide an additional tool for diagnostic management. Full article

Fungal infections are an increasingly growing public health concern, and Cryptococcus is one of the most problematic fungal organisms causing substantial mortality and morbidity worldwide. Clinically, this high incidence of cryptococcosis is most commonly seen in immunocompromised patients, especially those who lack an adaptive T cell response, such as HIV/AIDS patients. However, patients with other underlying immunodeficiencies are also at an increased risk for cryptococcosis. The adaptive immune response, in particular the Th1/Th17 T-cell-mediated responses, to pulmonary Cryptococcus infections are required for host protection. Dendritic cells (DCs), encompassing multiple subsets identified to date, are recognized as the major professional antigen-presenting cell (APC) subset essential for the initiation and execution of T-cell immunity. Apart from their prominent role in orchestration of the adaptive arm of the immune defenses, DCs are fully armed cells from the innate immune system capable of the recognition, uptake, and killing of the fungal cells. Thus, DCs serve as a critical point for the endpoint outcomes of either fungal control or unrestrained fungal infection. Multiple studies have shown that DCs are required for anti-cryptococcal defense in the lungs. In addition, the role of DCs in Cryptococcus gattii infections is just starting to be elucidated. C. gattii has recently risen to prominence with multiple outbreaks in the US and Canada, demonstrating increased virulence in non-immunocompromised individuals. C. gattii infection fails to generate an inflammatory immune response or a protective Th1/Th17 T cell response, at least in part, through a lack of proper DC function. Here we summarize the multiple roles of DCs, including subsets of DCs in both mouse and human models, the roles of DCs during cryptococcal infection, and mechanisms by cryptococcal cells to attempt to undermine these host defenses. Full article

Cryptococcosis is a systemic mycosis that causes pneumonia and meningoencephalitis. Strongyloidiasis is a chronic gastrointestinal infection caused by parasites of the genus Strongyloides. Cryptococcosis and strongyloidiasis affect the lungs and are more prevalent in the same world regions, i.e., Africa and tropical countries such as Brazil. It is undeniable that those coincidences may lead to the occurrence of coinfections. However, there are no studies focused on the interaction between Cryptococcus spp. and Strongyloides spp. In this work, we aimed to investigate the interaction between Strongyloides venezuelensis (Sv) and Cryptococcus gattii (Cg) in a murine coinfection model. Murine macrophage exposure to Sv antigens reduced their ability to engulf Cg and produce reactive oxygen species, increasing the ability of fungal growth intracellularly. We then infected mice with both pathogens. Sv infection skewed the host’s response to fungal infection, increasing lethality in a murine coinfection model. In addition to increased NO levels and arginase activity, coinfected mice presented a classic Th2 anti-Sv response: eosinophilia, higher levels of alternate activated macrophages (M2), increased concentrations of CCL24 and IL-4, and lower levels of IL-1β. This milieu favored fungal growth in the lungs with prominent translocation to the brain, increasing the host’s tissue damage. In conclusion, our data shows that primary Sv infection promotes Th2 bias of the pulmonary response to Cg-infection and worsens its pathological outcomes. Full article

Data about the relationship between their molecular types, virulence factors, clinical presentation, antifungal susceptibility profile, and outcome are still limited for Cryptococcus deuterogattii. This study aimed to evaluate the molecular and phenotypic characteristics of 24 C. deuterogattii isolates from the southeast region of Brazil. The molecular characterization was performed by multilocus sequence typing (MLST). The antifungal susceptibility profile was obtained according to CLSI-M27-A3 and EUCAST-EDef 7.1 methods. The virulence factors were evaluated using classic techniques. The isolates were divided into four populations. The molecular analysis suggests recombinant events in most of the groups evaluated. Resistance and susceptibility dose-dependent to fluconazole were evidenced in four isolates (16%) by EUCAST and in four isolates (16%) by CLSI methods. The agreement at ±two dilutions for both methods was 100% for itraconazole, ketoconazole, and voriconazole, 96% for amphotericin B, and 92% for fluconazole. Significant differences in virulence factor expression and antifungal susceptibility to itraconazole and amphotericin B were found. The mixed infection could be suggested by the presence of variable sequence types, differences in virulence factor production, and decreased antifungal susceptibility in two isolates from the same patient. The data presented herein corroborate previous reports about the molecular diversity of C. deuterogattii around the world. Full article

Anticytokine autoantibodies (ACAAs) can cause adult onset immunodeficiencies which mimic primary immunodeficiencies and can present as refractory and severe fungal infections. This paper provides an overview of the role of innate immunity, including key cytokines, in fungal infections and then describes four clinical scenarios where ACAAs are associated with severe presentations of a fungal infection: (1) Talaromyces marneffei infection and anti-interferon-γ, (2) histoplasmosis and anti-interferon-γ, (3) Cryptococcus gattii infection and anti-GM-CSF, and (4) mucocutaneous candidiasis and anti-IL-17A/F (IL-22). Testing for ACAAs and potential therapeutic options are discussed. Full article

Cryptococcus gattii is a pathogenic yeast, member of the C. neoformans/gattii complex. Previous work from our laboratory has established the presence of C. gattii on cacti lesions, providing proof that it can grow in a stressful environment. However, it is not known which part of the cactus the yeast uses for nutrients. The purpose of this research is to determine the ability of C. gattii to grow in different parts of the cactus to assess how the yeast adapts to grow in this unique environment. Cactus media were developed using the outer, inner, and whole cactus from Pilosocereus spp. Cryptcoccus gattii was grown on the different cactus media, along with potato dextrose agar as a control for 24 and 48 h at 30 °C. Compared to the control medium, yeast growth was reduced in all cactus media, while an increase in the capsule development of the yeast grown in the inner part and the whole-cactus media was observed. Interestingly, the yeast produces melanin when grown in the outer membrane medium, which was dependent on laccase, suggesting that the outer membrane may contain a precursor that stimulatates pigment production. To our knowledge, this is the first study addressing these key differences in the growth of C. gattii on different parts of the cactus. Full article

The species complexes Cryptococcus neoformans and Cryptococcus gattii are the causative agents of cryptococcosis. Virulence and susceptibility to antifungals may vary within each species according to the fungal genotype. Therefore, specific and easily accessible molecular markers are required to distinguish cryptic species and/or genotypes. Group I introns are potential markers for this purpose because they are polymorphic concerning their presence and sequence. Therefore, in this study, we evaluated the presence of group I introns in the mitochondrial genes cob and cox1 in different Cryptococcus isolates. Additionally, the origin, distribution, and evolution of these introns were investigated by phylogenetic analyses, including previously sequenced introns for the mtLSU gene. Approximately 80.5% of the 36 sequenced introns presented homing endonucleases, and phylogenetic analyses revealed that introns occupying the same insertion site form monophyletic clades. This suggests that they likely share a common ancestor that invaded the site prior to species divergence. There was only one case of heterologous invasion, probably through horizontal transfer to C. decagattii (VGIV genotype) from another fungal species. Our results showed that the C. neoformans complex has fewer introns compared to C. gattii. Additionally, there is significant polymorphism in the presence and size of these elements, both among and within genotypes. As a result, it is impossible to differentiate the cryptic species using a single intron. However, it was possible to differentiate among genotypes within each species complex, by combining PCRs of mtLSU and cox1 introns, for C. neoformans species, and mtLSU and cob introns for C. gattii species. Full article

Neuromeningeal cryptococcosis is a life-threatening infection of the central nervous system, caused by encapsulated yeast belonging to the Cryptococcus neoformans and Cryptococcus gattii species complexes. Recent data showed that virulence and antifungal resistance are variable for yeasts belonging to the C. gattii species complex. There is an increase in resistance to fluconazole for yeasts of the C. gattii species complex and the virulence is variable according to the genotype. In the present study, (i) we explored and compared the mechanisms of resistance to fluconazole between C. deuterogattii clinically resistant strains and induced fluconazole-resistant strains by exposure to fluconazole in vitro, and (ii) we studied their virulence in the Galleria mellonella study model. We demonstrated that the fluconazole resistance mechanisms involved were different between clinically resistant strains and induced resistant strains. We also demonstrated that fluconazole-induced resistant strains are less virulent when compared to the original susceptible strains. On the contrary, the clinically resistant strain tested maintains its virulence compared to fluconazole-susceptible strains of the same sequence type. Full article

When method-dependent categorical endpoints are available, namely either BPs or ECVs, MICs could aid in selecting the best treatment agent(s). BPs can categorize an isolate as either susceptible or resistant while the ECVs/ECOFFs can distinguish the wild type (WT, no known resistance mechanisms) from the Non-WT (NWT, harboring resistant mechanisms). Our literature review focused on the Cryptococcus species complex (SC) and the available methods and categorization endpoints. We also covered the incidence of these infections as well as the numerous Cryptococcus neoformans SC and C. gattii SC genotypes. The most important agents to treat cryptococcal infections are fluconazole (widely used), amphotericin B, and flucytosine. We provide data from the collaborative study that defined CLSI fluconazole ECVs for the most common cryptococcal species or genotypes and modes. EUCAST ECVs/ECOFFs are not yet available for fluconazole. We have summarized the incidence of cryptococccal infections (2000–2015) where fluconazole MICs were obtained by reference and commercial antifungal susceptibility tests. This occurrence is documented all over the world and those fluconazole MICs are mostly categorized by available CLSI ECVs/BPs as “resistant” instead of non-susceptible strains, including those by the commercial methods. As expected, the agreement between the CLSI and commercial methods is variable because SYO and Etest data could yield low/variable agreement (<90%) versus the CLSI method. Therefore, since BPs/ECVs are species and method dependent, why not gather sufficient MICs by commercial methods and define the required ECVs for these species? Full article

Background: Cross-resistance to medical azoles by exposure to azole pesticides is well documented for Aspergillus family fungi but is poorly evaluated for other environmental pathogen fungi, particularly for yeasts belonging to the Cryptococcus neoformans/Cryptococcus gattii species complexes. Methods: One thousand C. neoformans yeast were exposed to various concentrations of seven different commonly used azole pesticides. Clones surviving exposure were picked randomly, and their minimal inhibitory concentrations (MICs) of fluconazole, voriconazole, posaconazole, itraconazole and isavuconazole were assessed. Results: Depending on the pesticide used for exposure, up to 13.3% of selected Cryptococcus colonies showed a phenotype of resistance to fluconazole, and among them, several showed cross-resistance to another or several other medical azoles. Molecular mechanisms involved in the resistance setups seem to be dependent on ERG11 and AFR1 gene overexpression. Conclusion: Exposure to any of the seven azole pesticides tested is capable of increasing the MIC of fluconazole in C. neoformans, including up to the level of the fluconazole-resistant phenotype, as well as generating cross-resistance to other medical azoles in some cases. Full article