Cryptococcus gattii in North American Pacific Northwest: Whole-Population Genome Analysis Provides Insights into Species Evolution and Dispersal

The emergence of distinct populations of Cryptococcus gattii in the temperate North American Pacific Northwest (PNW) was surprising, as this species was previously thought to be confined to tropical and semitropical regions. Beyond a new habitat niche, the dominant emergent population displayed increased virulence and caused primary pulmonary disease, as opposed to the predominantly neurologic disease seen previously elsewhere. Whole-genome sequencing was performed on 118 C. gattii isolates, including the PNW subtypes and the global diversity of molecular type VGII, to better ascertain the natural source and genomic adaptations leading to the emergence of infection in the PNW. Overall, the VGII population was highly diverse, demonstrating large numbers of mutational and recombinational events; however, the three dominant subtypes from the PNW were of low diversity and were completely clonal. Although strains of VGII were found on at least five continents, all genetic subpopulations were represented or were most closely related to strains from South America. The phylogenetic data are consistent with multiple dispersal events from South America to North America and elsewhere. Numerous gene content differences were identified between the emergent clones and other VGII lineages, including genes potentially related to habitat adaptation, virulence, and pathology. Evidence was also found for possible gene introgression from Cryptococcus neoformans var. grubii that is rarely seen in global C. gattii but that was present in all PNW populations. These findings provide greater.IMPORTANCE Cryptococcus gattii emerged in the temperate North American Pacific Northwest (PNW) in the late 1990s. Beyond a new environmental niche, these emergent populations displayed increased virulence and resulted in a different pattern of clinical disease. In particular, severe pulmonary infections predominated in contrast to presentation with neurologic disease as seen previously elsewhere. We employed population-level whole-genome sequencing and analysis to explore the genetic relationships and gene content of the PNW C. gattii populations. We provide evidence that the PNW strains originated from South America and identified numerous genes potentially related to habitat adaptation, virulence expression, and clinical presentation. Characterization of these genetic features may lead to improved diagnostics and therapies for such fungal infections. The data indicate that there were multiple recent introductions of C. gattii into the PNW. Public health vigilance is warranted for emergence in regions where C. gattii is not thought to be endemic

Primeiro registro de Cryptococcus neoformans em excretas de pombos provenientes de locais públicos e residenciais de área metropolitana de Cuiabá, Estado do Mato Grosso, Brasil

RESUMO A criptococose é micose sistêmica potencialmente grave causada por duas espécies do gênero Cryptococcus que acometem tanto homens como animais: Cryptococcus neoformans e C. gattii. São infecções cosmopolitas e emergentes, resultantes da interação do hospedeiro - humano e animal versus meio ambiente. A proposta deste trabalho foi avaliar a ocorrência de C. neoformans em 122 amostras de excretas secas de pombos coletadas em 49 locais na cidade de Cuiabá, Estado do Mato Grosso, Brasil, incluindo: praças públicas (n = 5), igrejas (n = 4), instituições de ensino (n = 3), unidades de saúde (n = 8), áreas abertas exibindo cobertura de amianto (n = 4), conjuntos residenciais domiciliares (n = 23), uma fábrica (n = 1) e um presídio (n = 1). Semeadura de suspensão de amostras em meio ágar niger (NSA), identificação fenotípica por provas bioquímicas e teste em meio de canavanina-glicina-azul de bromotimol, das colônias isoladas com pigmentação marrom escura. Foi também utilizada a técnica da reação em cadeia da polimerase com pares de iniciadores específicos para identificação de C. neoformans. As amostras foram coletadas de julho a dezembro de 2010. Cryptococcus neoformans foi isolado em oito (6,6%) de 122 amostras correspondendo a seis (12,2%) dos 49 sítios analisados. Cryptococcus neoformans associado a excretas de pombos ocorre em áreas de Cuiabá, predominando em residências nas amostras analisadas, constituindo fator de risco potencial para aquisição da doença tanto para indivíduos imunocomprometidos como imunocompetentes.SUMMARY Cryptococcosis is a severe systemic mycosis caused by two species of Cryptococcus that affect humans and animals: C. neoformans and C. gattii. Cosmopolitan and emergent, the mycosis results from the interaction between a susceptible host and the environment. The occurrence of C. neoformans was evaluated in 122 samples of dried pigeon excreta collected in 49 locations in the City of Cuiabá, State of Mato Grosso, Brazil, including public squares (n = 5), churches (n = 4), educational institutions (n = 3), health units (n = 8), open areas covered with asbestos (n = 4), residences (n = 23), factory (n = 1) and a prison (n = 1). Samples collected from July to December of 2010 were seeded on Niger seed agar (NSA). Dark brown colonies were identified by urease test, carbon source assimilation tests and canavanine-glycine-bromothymol blue medium. Polymerase chain reaction primer pairs specific for C. neoformans were also used for identification. Cryptococcus neoformans associated to pigeon excreta was isolated from eight (6.6%) samples corresponding to six (12.2%) locations. Cryptococcus neoformans was isolated from urban areas, predominantly in residences, constituting a risk of acquiring the disease by immunocompromised and immunocompetent individuals

MALDI-TOF MS Enables the Rapid Identification of the Major Molecular Types within the Cryptococcus neoformans/C. gattii Species Complex

BACKGROUND: The Cryptococcus neoformans/C. gattii species complex comprises two sibling species that are divided into eight major molecular types, C. neoformans VNI to VNIV and C. gattii VGI to VGIV. These genotypes differ in host range, epidemiology, virulence, antifungal susceptibility and geographic distribution. The currently used phenotypic and molecular identification methods for the species/molecular types are time consuming and expensive. As Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) offers an effective alternative for the rapid identification of microorganisms, the objective of this study was to examine its potential for the identification of C. neoformans and C. gattii strains at the intra- and inter-species level. METHODOLOGY: Protein extracts obtained via the formic acid extraction method of 164 C. neoformans/C. gattii isolates, including four inter-species hybrids, were studied. RESULTS: The obtained mass spectra correctly identified 100% of all studied isolates, grouped each isolate according to the currently recognized species, C. neoformans and C. gattii, and detected potential hybrids. In addition, all isolates were clearly separated according to their major molecular type, generating greater spectral differences among the C. neoformans molecular types than the C. gattii molecular types, most likely reflecting a closer phylogenetic relationship between the latter. The number of colonies used and the incubation length did not affect the results. No spectra were obtained from intact yeast cells. An extended validated spectral library containing spectra of all eight major molecular types was established. CONCLUSIONS: MALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic Cryptococcus species and offers a simple method for the separation of the eight major molecular types and the detection of hybrid strains within this species complex in the clinical laboratory. The obtained mass spectra provide further evidence that the major molecular types warrant variety or even species status

Cryptococcus gattii in North American Pacific Northwest: whole-population genome analysis provides insights into species evolution and dispersal

The emergence of distinct populations of Cryptococcus gattii in the temperate North American Pacific Northwest (PNW) was surprising, as this species was previously thought to be confined to tropical and semitropical regions. Beyond a new habitat niche, the dominant emergent population displayed increased virulence and caused primary pulmonary disease, as opposed to the predominantly neurologic disease seen previously elsewhere. Whole-genome sequencing was performed on 118 C. gattii isolates, including the PNW subtypes and the global diversity of molecular type VGII, to better ascertain the natural source and genomic adaptations leading to the emergence of infection in the PNW. Overall, the VGII population was highly diverse, demonstrating large numbers of mutational and recombinational events; however, the three dominant subtypes from the PNW were of low diversity and were completely clonal. Although strains of VGII were found on at least five continents, all genetic subpopulations were represented or were most closely related to strains from South America. The phylogenetic data are consistent with multiple dispersal events from South America to North America and elsewhere. Numerous gene content differences were identified between the emergent clones and other VGII lineages, including genes potentially related to habitat adaptation, virulence, and pathology. Evidence was also found for possible gene introgression from Cryptococcus neoformans var. grubii that is rarely seen in global C. gattii but that was present in all PNW populations. These findings provide greater understanding of C. gattii evolution in North America and support extensive evolution in, and dispersal from, South America. Importance: Cryptococcus gattii emerged in the temperate North American Pacific Northwest (PNW) in the late 1990s. Beyond a new environmental niche, these emergent populations displayed increased virulence and resulted in a different pattern of clinical disease. In particular, severe pulmonary infections predominated in contrast to presentation with neurologic disease as seen previously elsewhere. We employed population-level whole-genome sequencing and analysis to explore the genetic relationships and gene content of the PNW C. gattii populations. We provide evidence that the PNW strains originated from South America and identified numerous genes potentially related to habitat adaptation, virulence expression, and clinical presentation. Characterization of these genetic features may lead to improved diagnostics and therapies for such fungal infections. The data indicate that there were multiple recent introductions of C. gattii into the PNW. Public health vigilance is warranted for emergence in regions where C. gattii is not thought to be endemic

MLST reveals a clonal population structure for Cryptococcus neoformans molecular type VNI isolates from clinical sources in Amazonas, Northern-Brazil

Cryptococcosis is considered endemic in Amazonas state, occurring more frequently in individuals with AIDS, who are predominantly infected by Cryptococcus neoformans molecular type VNI. Infections by Cryptococcus gattii VGII predominate in immunocompetent hosts from the American continent and are associated with outbreaks in North America, particularly the subtypes VGIIa and VGIIb, which are also present in the Brazilian Amazon region. Despite few environmental studies, several aspects of the molecular epidemiology of this disease in Amazonas remain unclear, including the limited use of multilocus sequence typing (MLST) to evaluate the genetic population structure of clinical isolates, mainly C. neoformans. Therefore, we used MLST to identify the sequence types of 38 clinical isolates of C. neoformans VNI and C. gattii VGII and used phylogenetic analysis to evaluate their genetic relationship to global isolates. Records of 30 patients were analyzed to describe the current scenario of cryptococcosis in the region and their associations with the different subtypes. Broth microdilution was also performed to determine the susceptibility profile to the antifungals amphotericin B, fluconazole and itraconazole. MLST identified that patients with HIV (n = 26) were exclusively affected by VNI strains with ST93, and among the VGII strains (n = 4), three STs (ST5, ST172 and the new ST445) were identified. An in-hospital lethality of 54% was observed in the HIV group, and there were no significant differences in the clinical aspects of the disease between the HIV and non-HIV groups of patients. In addition, all isolates were susceptible to the antifungals tested. Therefore, in Amazonas state, VNI isolates are a genetically monotypic group, with ST93 being highly important in HIV individuals. © 2018 Rocha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Altimetry for the future: Building on 25 years of progress

In 2018 we celebrated 25 years of development of radar altimetry, and the progress achieved by this methodology in the fields of global and coastal oceanography, hydrology, geodesy and cryospheric sciences. Many symbolic major events have celebrated these developments, e.g., in Venice, Italy, the 15th (2006) and 20th (2012) years of progress and more recently, in 2018, in Ponta Delgada, Portugal, 25 Years of Progress in Radar Altimetry. On this latter occasion it was decided to collect contributions of scientists, engineers and managers involved in the worldwide altimetry community to depict the state of altimetry and propose recommendations for the altimetry of the future. This paper summarizes contributions and recommendations that were collected and provides guidance for future mission design, research activities, and sustainable operational radar altimetry data exploitation. Recommendations provided are fundamental for optimizing further scientific and operational advances of oceanographic observations by altimetry, including requirements for spatial and temporal resolution of altimetric measurements, their accuracy and continuity. There are also new challenges and new openings mentioned in the paper that are particularly crucial for observations at higher latitudes, for coastal oceanography, for cryospheric studies and for hydrology. The paper starts with a general introduction followed by a section on Earth System Science including Ocean Dynamics, Sea Level, the Coastal Ocean, Hydrology, the Cryosphere and Polar Oceans and the ‘‘Green” Ocean, extending the frontier from biogeochemistry to marine ecology. Applications are described in a subsequent section, which covers Operational Oceanography, Weather, Hurricane Wave and Wind Forecasting, Climate projection. Instruments’ development and satellite missions’ evolutions are described in a fourth section. A fifth section covers the key observations that altimeters provide and their potential complements, from other Earth observation measurements to in situ data. Section 6 identifies the data and methods and provides some accuracy and resolution requirements for the wet tropospheric correction, the orbit and other geodetic requirements, the Mean Sea Surface, Geoid and Mean Dynamic Topography, Calibration and Validation, data accuracy, data access and handling (including the DUACS system). Section 7 brings a transversal view on scales, integration, artificial intelligence, and capacity building (education and training). Section 8 reviews the programmatic issues followed by a conclusion