COVID-19–Associated Pulmonary Aspergillosis

Disseminated sporotrichosis occurs in individuals with impaired cellular immunity, such as in cases of neoplasia, transplantation, diabetes, and especially, acquired immunodeficiency syndrome. This report presents a 32-year-old Brazilian human immunodeficiency virus (HIV)-infected patient who developed a protracted condition of disseminated sporotrichosis with endocarditis, bilateral endophthalmitis, and lymphatic involvement. He needed cardiac surgery to replace the mitral valve. Sporothrix brasiliensis isolates were recovered from cultures of subcutaneous nodules and mitral valve fragments. Species identification was based on classical and molecular methods. The patient received amphotericin B for 52 days and subsequently, oral itraconazole. He remains asymptomatic, and he is on maintenance therapy with itraconazole. Despite his positive clinical outcome, he developed bilateral blindness. To our knowledge, this case is the first report of endocarditis and endophthalmitis caused by S. brasiliensis.

Galleria mellonella larvae have been widely used as alternative non-mammalian models for the study of fungal virulence and pathogenesis. The larvae can be acquired in small volumes from worm farms, pet stores, or other independent suppliers commonly found in the United States and parts of Europe. However, in countries with no or limited commercial availability, the process of shipping these larvae can cause them stress, resulting in decreased or altered immunity. Furthermore, the conditions used to rear these larvae including diet, humidity, temperature, and maintenance procedures vary among the suppliers. Variation in these factors can affect the response of G. mellonella larvae to infection, thereby decreasing the reproducibility of fungal virulence experiments. There is a critical need for standardized procedures and incubation conditions for rearing G. mellonella to produce quality, unstressed larvae with the least genetic variability. In order to standardize these procedures, cost-effective protocols for the propagation and maintenance of G. mellonella larvae using an artificial diet, which has been successfully used in our own laboratory, requiring minimal equipment and expertise, are herein described. Examples for the application of this model in fungal pathogenicity and gene knockout studies as feasible alternatives for traditionally used animal models are also provided.

Cryptococcosis is an important fungal infection in immunocompromised individuals, especially those infected with HIV. In Brazil, despite the free availability of antiretroviral therapy (ART) in the public health system, the mortality rate due to Cryptococcus neoformans meningitis is still high. To obtain a more detailed picture of the population genetic structure of this species in southeast Brazil, we studied 108 clinical isolates from 101 patients and 35 environmental isolates. Among the patients, 59% had a fatal outcome mainly in HIV-positive male patients. All the isolates were found to be C. neoformans var. grubii major molecular type VNI and mating type locus alpha. Twelve were identified as diploid by flow cytometry, being homozygous (AαAα) for the mating type and by PCR screening of the STE20, GPA1, and PAK1 genes. Using the ISHAM consensus multilocus sequence typing (MLST) scheme, 13 sequence types (ST) were identified, with one being newly described. ST93 was identified from 81 (75%) of the clinical isolates, while ST77 and ST93 were identified from 19 (54%) and 10 (29%) environmental isolates, respectively. The southeastern Brazilian isolates had an overwhelming clonal population structure. When compared with populations from different continents based on data extracted from the ISHAM-MLST database (mlst.mycologylab.org) they showed less genetic variability. Two main clusters within C. neoformans var. grubii VNI were identified that diverged from VNB around 0.58 to 4.8 million years ago. The members of the Cryptococcus neoformans / Cryptococcus gattii species complex are the cause of cryptococcosis, a life-threatening human disease responsible for 624,000 deaths annually. Infection is acquired through inhalation of dehydrated yeast cells from environmental sources. After reaching the lungs, the fungus disseminates to the central nervous system causing meningoencephalitis. The majority of meningitis cases in HIV-infected patients are caused by C. neoformans, a species well studied in regions with a high prevalence of HIV infection, such as Asia and Africa. A similar high prevalence has been reported from Brazil however the epidemiology of these infections is less well understood. We studied clinical and environmental isolates from the southeast region of Brazil using MLST. The results that we obtained showed a clonal population structure of C. neoformans var. grubii VNI, with low variability when compared against populations from different continents. This lower variability is probably the result of multiple recent dispersal events from Africa to the Americas. The majority of clinical isolates were of one sequence type (ST93), which was also found in environmental samples. By expanding the analysis to isolates from around the globe, it was possible to identify two major groups among C. neoformans var. grubii VNI.

Cryptococcus neoformans and Cryptococcus gattii are responsible globally for almost one million cryptococcosis cases yearly, mostly in immunocompromised patients, such as those living with HIV. Infections due to C. gattii have mainly been described in tropical and subtropical regions, but its adaptation to temperate regions was crucial in the species evolution and highlighted the importance of this pathogenic yeast in the context of disease. Cryptococcus gattii molecular type VGII has come to the forefront in connection with an on-going emergence in the Pacific North West of North America. Taking into account that previous work pointed towards South America as an origin of this species, the present work aimed to assess the genetic diversity within the Brazilian C. gattii VGII population in order to gain new insights into its origin and global dispersal from the South American continent using the ISHAM consensus MLST typing scheme. Our results corroborate the finding that the Brazilian C. gattii VGII population is highly diverse. The diversity is likely due to recombination generated from sexual reproduction, as evidenced by the presence of both mating types in clinical and environmental samples. The data presented herein strongly supports the emergence of highly virulent strains from ancestors in the Northern regions of Brazil, Amazonia and the Northeast. Numerous genotypes represent a link between Brazil and other parts of the world reinforcing South America as the most likely origin of the C. gattii VGII subtypes and their subsequent global spread, including their dispersal into North America, where they caused a major emergence. Cryptococcus neoformans and Cryptococcus gattii are fungal agents responsible globally for almost one million cryptococcosis cases yearly, mostly in immunocompromised patients, such as those living with HIV. Cryptococcosis is a life-threatening mycosis, frequently causing meningoencephalitis. Infections due to C. gattii were originally described in tropical and subtropical regions, but its adaptation to temperate regions was highlighted by the emergence in the Pacific North West of North America by C. gattii molecular type VGII. The present work aimed to assess the genetic diversity within the Brazilian C. gattii VGII population to gain new insights into its origin and global dispersal from the South American continent using the ISHAM MLST consensus typing scheme. Our results corroborate that the Brazilian C. gattii VGII population is highly diverse, and strongly supports the emergence of highly virulent strains from ancestors in the Northern regions of Brazil. Numerous genotypes represent a link between Brazil and other parts of the world, and the isolates from the transitional ecological area in Northeast Brazil are the most likely ancestor lineages, translocating from caatinga/cerrado by adapting progressively throughout Amazonia in South America, and spread to the North American Pacific Northwest region and other parts of the world on multiple occasions.