Clonal dispersal of Cryptococcus gattii VGII in an endemic region of cryptococcosis in Colombia

This study characterized the genotype and phenotype of Cryptococcus gattii VGII isolates from Cucuta, an endemic region of cryptococcal disease in Colombia, and compared these traits with those from representative isolates from the Vancouver Island outbreak (VGIIa and VGIIb). Genetic diversity was assessed by multilocus sequence typing (MLST) analysis. Phenotypic characteristics, including growth capacity under different temperature and humidity conditions, macroscopic and microscopic morphology, phenotypic switching, mating type, and activity of extracellular enzymes were studied. Virulence was studied in vivo in a mouse model. MLST analysis showed that the isolates from Cucuta were highly clonal, with ST25 being the most common genotype. Phenotypically, isolates from Cucuta showed large cell and capsular sizes, and shared phenotypic traits and enzymatic activities among them. The mating type a prevailed among the isolates, which were fertile and of considerable virulence in the animal model. This study highlights the need for a continuous surveillance of C. gattii in Colombia, especially in endemic areas like Cucuta, where the highest number of cryptococcosis cases due to this species is reported. This will allow the early detection of potentially highly virulent strains that spread clonally, and can help prevent the occurrence of outbreaks in Colombia and elsewhere. © 2019 by the authors

Indoor Dust as a Source of Virulent Strains of the Agents of Cryptococcosis in the Rio Negro Micro-Region of the Brazilian Amazon.

Cryptococcosis, a potentially fatal mycosis in humans, is acquired via exposure to exogenous environmental sources. This study aimed to investigate the frequency, genetic diversity, and virulence of cryptococcal strains isolated from indoor dust in the Rio Negro micro-region of the Brazilian Amazon. A total of 8.9% of the studied houses were positive, recovering nine Cryptococcus neoformans VNI and 16 C. gattii VGII isolates, revealing an endemic pattern in domestic microenvironments. The International Society for Human and Animal Mycology (ISHAM) consensus multilocus sequence typing (MLST) scheme for the C. neoformans/C. gattii species complexes identified two sequence types (STs), ST93 and ST5, amongst C. neoformans isolates and six STs amongst C. gattii isolates, including the Vancouver Island Outbreak ST7 (VGIIa) and ST20 (VGIIb), the Australian ST5, and ST264, ST268 and ST445, being unique to the studied region. Virulence studies in the Galleria mellonella model showed that five C. gattii strains and one C. neoformans strain showed a similar pathogenic potential to the highly virulent Vancouver Island outbreak strain CDR265 (VGIIa). The findings of this study indicate that humans can be exposed to the agents of cryptococcosis via house dust, forming the basis for future studies to analyze the impact of early and continuous exposure to indoor dust on the development of subclinical or clinical infections

Characterization of the human pathogenic species Cryptococcus neoformans and Cryptococcus gattii with a special emphasis on emerging molecular types within C. gattii

The rising incidence of cryptococcosis, a life-threatening fungal infection affecting both immunocompromised and immunocompetent humans and animals, along with the emergence of outbreaks of infection, highlight the need of an increased and constant vigilance of its etiological agents Cryptococcus neoformans and C. gattii. The initial part of this PhD thesis evaluates MALDI-TOF MS and hyperbranched rolling circle PCR as two new alternative methodologies for the rapid and accurate identification of these two species and their major molecular types. The second part shows the utility of the insects Drosophila melanogaster and Galleria mellonella as invertebrate models of infection to study cryptococcal pathogenesis and virulence factors and to screen for strains with different levels of virulence. In the last part, different molecular approaches are applied to understand the epidemiology of cryptococcosis, and the differences regarding the genetic diversity, virulence and antifungal susceptibility between and within major molecular types/species. Being part of an ongoing research collaboration among different institutions, that continuously study the epidemiology, genetics and pathogenesis of these medically important yeasts, the population studies conducted during this PhD strongly contribute to our knowledge about the regional and global spread of Cryptococcus and the epidemiology of cryptococcosis

Multi-locus sequence typing and phylogenetics of Cryptococcus neoformans AD hybrids

Hybrid AD strains of the human pathogenic Cryptococcus neoformans species complex have been reported from many parts of the world. However, their origin, diversity, and evolution are incompletely understood. In this study, we analyzed 102 AD hybrid strains representing 21 countries on five continents. For each strain, we obtained its mating type and its allelic sequences at each of the seven loci that have been used for genotyping haploid serotypes A and D strains of the species complex by the Cryptococcus research community. Our results showed that most AD hybrids exhibited loss of heterozygosity at one or more of the seven analyzed loci. Phylogenetic and population genetic analyses of the allelic sequences revealed multiple origins of the hybrids within each continent, dating back to one million years ago in Africa and up to the present in other continents. We found evidence for clonal reproduction and long-distance dispersal of these hybrids in nature. Comparisons with the global haploid serotypes A and D strains identified new alleles and new haploid multi-locus genotypes in AD hybrids, consistent with the presence of yet-to-be discovered genetic diversity in haploid populations of this species complex in nature. Together, our results indicate that AD hybrids can be effectively genotyped using the same multi-locus sequencing type approach as that established for serotypes A and D strains. Our comparisons of the AD hybrids among each other as well as with the global haploid serotypes A and D strains revealed novel genetic diversity as well as evidence for multiple origins and dynamic evolution of these hybrids in nature

Underrated and deadly! So are invasive fungal infections

En el reino de estos gérmenes existen alrededor de seis millones de especies, pero tres en particular afectan la salud humana al punto de convertirse en una amenaza para la salud pública y un duro desafío para tratamientos que salvarían vidas.In the kingdom of these germs there are around six million species, but three in particular affect human health to the point of becoming a threat to public health and a tough challenge for life-saving treatments

Molecular techniques used in the diagnosis and identification of fungi

El éxito de las técnicas moleculares para el diagnóstico de enfermedades causadas por virus y bacterias( aeróbicas, anaeróbicas y micobacterias) y para la identificación de sus agentes causales ha alentado a muchos laboratoristas y clínicos a explorar la función de dichas técnicas en el diagnóstico de infecciones fúngicas invasoras (IFI) y otras micosis, y su aplicación para la identificación de hongos patógenos. Dado que las IFI y otras micosis representan una causa importante de morbilidad y mortalidad en pacientes inmunocromprometidos y con otros factores de riesgo, técnicas de diagnóstico rápidas y robustas y la identificación correcta de sus agentes causales son cada vez más necesarias

Caracterización molecular de aislamientos invasores colombianos de Streptococcus pneumoniae serotipo 5, recuperados entre 1994 y 2004

Streptococcus pneumoniae serotipo 5 es una causa importante de enfermedad invasiva en Colombia. Estudios preliminares han demostrado la circulación de un clon reconocido por la red de epidemiología molecular de neumococo (PMEN) como clon 19-Colombia5, susceptible a penicilina pero asociado con resistencia a tetraciclina y cloranfenicol. Con el objetivo de establecer las relaciones genéticas de aislamientos invasores de S. pneumoniae serotipo 5 recuperados en Colombia entre 1994 y 2004, con el clon 19-Colombia5, se estudiaron 83 aislamientos, que tenían datos de susceptibilidad a penicilina, vancomicina, ceftriaxona, eritromicina, trimetoprim sulfametoxazol, cloranfenicol y tetraciclina, de los cuales, 29 fueron recuperados de niños menores de cinco años. Se les determinó el patrón de restricción por electroforesis de campo pulsado (PFGE), usando la enzima Sma I. La similitud genética entre los aislamientos y el clon se estableció según los criterios de Tenover utilizando el programa Fingerprinting‰ II para la generación de dendrogramas. Todos los aislamientos se agruparon en el patrón electroforético A, del cual se derivaron 18 subtipos, todos relacionados con el clon 19-Colombia5. El patrón A se estableció en 32 aislamientos (38,6%), el subtipo A8 en 18 (21,7%) y el A5 en 10 (12%), los patrones restantes agruparon los otros 23 aislamientos. Los 34 aislamientos resistentes a tetraciclina y cloranfenicol, estuvieron relacionados con el patrón electroforético A (n=32) y los subtipos A16 (n=1) y A28 (n=1), caracterizados por presentar una banda de 340 Kb, al igual que el clon. Estos resultados muestran la prevalencia y circulación continua de aislamientos de S. pneumoniae serotipo 5, los cuales están genéticamente relacionados con el clon 19-Colombia

Reduced Susceptibility to Azoles in Cryptococcus gattii Correlates with the Substitution R258L in a Substrate Recognition Site of the Lanosterol 14-?-Demethylase

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a lifethreatening fungal infection affecting mostly immunocompromised patients. In fact, cryptococcal meningitis accounts for about 19% of AIDS-related deaths in the world. Because of long-term azole therapies to treat this mycosis, resistance to fluconazole leading to treatment failure and poor prognosis has long been reported for both fungal species. Among the mechanisms implicated in resistance to azoles, mutations in the ERG11 gene, encoding the azole target enzyme lanosterol 14-a-demethylase, have been described. This study aimed to establish the amino acid composition of ERG11 of Colombian clinical isolates of C. neoformans and C. gattii and to correlate any possible substitution with the in vitro susceptibility profile of the isolates to fluconazole, voriconazole, and itraconazole. Antifungal susceptibility testing results showed that C. gattii isolates are less susceptible to azoles than C. neoformans isolates, which could correlate with differences in the amino acid composition and structure of ERG11 of each species. In addition, in a C. gattii isolate with high MICs for fluconazole (64 mg/mL) and voriconazole (1 mg/mL), a G973T mutation resulting in the substitution R258L, located in substrate recognition site 3 of ERG11, was identified. This finding suggests the association of the newly reported substitution with the azole resistance phenotype in C. gattii. Further investigations are needed to determine the exact role that R258L plays in the decreased susceptibility to fluconazole and voriconazole, as well as to determine the participation of additional mechanisms of resistance to azole drugs

Fungemia mortal por Cryptococcus albidus en una anciana diabética que presenta derrame pleural

This is a fatal case of fungaemia due to Cryptococcus albidus in an elderly woman with type 2 diabetes mellitus who presented pleural effusion as the only clinical presentation. Not only infections of the pleura are uncommon presentations of pulmonary cryptococcosis, but these infections due to non-C. neoformans species are extremely rare. This report places C. albidus on the growing number of disseminated mycosis-causing agents in diabetic patients