PLoS One. 2015 Feb 17;10(2):e0115866.

Environmental isolation of Cryptococcus gattii VGII from indoor dust associate to typical wooden houses in the deep Amazon of the Rio Negro basin

 

Fábio Brito-Santos1, Gláucia Barbosa1, Luciana Trilles1, Bodo Wanke1, Wieland Meyer1,2, Filipe Anibal Carvalho-Costa3, Marília Martins Nishikawa4, Márcia dos Santos Lazéra1

1Mycology Laboratory, National Institute of Infectious Diseases Evandro Chagas (INI), Fiocruz, Rio de Janeiro, Brazil; 2Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Sydney Medical School-Westmead Hospital, Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Westmead Millennium Institute, Sydney, NSW, Australia; 3Laboratory of Molecular Epidemiology and Systematics, Institute Oswaldo Cruz, FIOCRUZ; 4National Institute for Quality Control in Health, INCQS/FIOCRUZ, Rio de Janeiro, Brazil.

 

Abstract

Cryptococcosis is a human fungal infection of significant mortality and morbidity, especially when causing meningoencephalitis. The agents of cryptococcosis, C. neoformans and C. gattii, are found worldwide, and can be classified into eight major molecular types: VNI-VNIV and VGI-VGIV, respectively. Primary cryptococcosis caused by molecular type VGII (serotype B, MATalpha) prevails in immunocompetent patients in the North and Northeast of Brazil, revealing an endemic regional pattern. Since 1999, C. gattii VGII has been involved in an ongoing outbreak in Vancouver, Canada, which expanded subsequently into the Northwest of the United States, two temperate regions. Exposure to infectious propagules (desiccated yeasts or basidiospores), dispersed in the environment, related to various organic substrates, mainly decomposing wood in and around dwellings, initiates the infection process. The present study aimed to investigate the presence of the agents of cryptococcosis in dust from dwellings in the upper Rio Negro, in the municipality of Santa Isabel do Rio Negro, in the Brazilian state Amazonas. Indoor dust was collected from 51 houses, diluted and plated on bird-seed agar. Dark brown colonies were identified phenotypically and genotypically by URA5 restriction fragment length polymorphism analysis and multilocus sequence typing (MLST). The mating type was identified using pheromone-specific primers. Three of the 51 houses were positive for C. gattii molecular type VGII, MATα and MATa, showing a high prevalence of this agent. MLST studies identified eight subtypes, VGIIb (ST7), VGIIa (ST20), (ST5) and 5 new subtypes unique to the region. For the first time in the state of Amazonas, C. gattii VGII MATα and MATa were isolated from the environment, indicating Cryptococcus is endemic in this state. This is the first description of MLST subtypes on environmental isolates in the Brazilian Amazon, indicating domiciliary dust as a potential source for human infection with different subtypes of C. gattii VGII MATα and MATa.

PMID: 25688971

 

Supplement:

Cryptococcosis is an important infectious disease globally and, as an AIDS-associated disease, is the leading cause of death along with tuberculosis. Inhalation of airborne cryptococci initiates pulmonary infection, which may be asymptomatic or subclinical, unspecific and almost always regressive or may spread to other organs mainly the central nervous system (CNS) and cause meningitis. Disease is caused by the two sibling species Cryptococcus neoformans (molecular types VNI-VNIV) and Cryptococcus gattii (molecular types VGI-VGIV). The agents of cryptococcosis are found in decaying wood, trees, bird excreta, soil, water, and air. Exposure to those exogenous sources can lead to colonization and subsequent infection of humans and animals. Infection due to C. neoformans is distributed worldwide and attains mainly immunosuppressed hosts. Fatal infections like meningoencephalitis in apparently immunocompetent hosts are mostly caused by C. gattii, which has progressively expanded its natural range from tropical to temperate areas around the world, showing a great potential to generate outbreaks due to different virulent variants. Both species are endemic in the North and Northeastern Brazil, as indicated by the high numbers of infected children and adolescents (20 up to 30 %), which suggest early infection in native hosts (1).

 

figure 1-3

Figure 1. (1a) Wooden houses in Santa Isabel do Rio Negro from which C. gattii was collected. (1b) Sample collection from wooden electricity poles in the same locality from which C. gattii and C. neoformans have been isolated.

 

We hypothesize humans are exposed daily to the infectious propagules in the Brazilian Amazon region. To demonstrate this, we conducted an environmental study analyzing indoor dust from the houses (Figure 1a) of inhabitants and wooden electricity poles (Figure 1b) of the city Santa Isabel of Rio Negro, located in the upper Rio Negro river in the Amazonas state, Brazil. Indoor dust was collected from 51 typical wooden houses. Three (5.9%) houses were positive, yielding uncountable colonies of C. gattii molecular type VGII (Figure 2). From the obtained colonies, 45 were selected for further molecular analysis. Surprisingly eight MLST subtypes of VGII (ST5, ST7, ST20, ST264, ST265, ST266, ST267 and ST268) were identified in the small area studied (approximately 5 km2 in Amazonia), the highest genetic diversity described to date in environmental studies worldwide (2). The diversity of C. gattii VGII found inside the wooden houses in a countryside town in the amazon is an important finding since it may point to a much broader impact on human health, considering the human occupation with the construction of rustic wooden houses in recently deforested areas in the last decades. Interestingly around the positive houses, all the native trees have been cut down during and after the settlement. Later on trunks of native trees were used as wooden poles throughout the city to support the electric wires. From 10 wooden poles, 2 decomposing wood samples were positive for the agents of cryptococcosis, molecular types VGII (C. gattii) and VNI (C. neoformans). Our findings show that the residents of Santa Isabel do Rio Negro live daily with the presence of the agents of cryptococcosis, C. gattii VGII or C. neoformans VNI. We believe that the same happens in other cities with a similar occupation profile throughout Amazonia. Both species responsible for cryptococcosis seem to be well adapted to environmental conditions found in Amazonia, which is drastically transformed by human environmental intervention, exposing people to potential pathogens. Destroyed environments lead to the loss of microbial biodiversity and new competition, favoring overgrowth of the more adapted species. In this specific case, C. gattii and C. neoformans, being wood decomposers, are very well adapted to environmental conditions in the villages, which are basically built with woods from the rainforest. A study in the wild tropical forest without anthropic action in Amazonia demonstrated the presence of C. gattii inhabiting a Guettarda tree, suggesting the original ecological niche of the species (3). In urban areas, the prevalence of both species is much higher, increasing the potential risk of human exposure to those pathogens. Due to their proximity to human dwellings, it is highly possible that a phase transition and adaptation of these agents may occur in the wooden electricity poles, thus allowing a secondary dispersion to the home environments and, as such, they function as true biological corridors. Interestingly, some of the in Santa Isabel do Rio Negro identified subtypes (ST7 (VGIIb), ST20 (VGIIa)) are also responsible for the ongoing outbreak in the Pacific Northwest (Canada and United States) (4, 5). Recent studies of coalescence gene genealogy and whole genome analysis on the origin of the geographically expanding C. gattii VGII population have demonstrate that these outbreak strains have arisen from a highly-recombining C. gattii population in the pristine environment of the native rainforest of Northern Brazil (4, 5).

The importance of this study.

This study, for the first time, found VGIIa and VGIIb in the state of Amazonas, where they were isolated from the environment, specifically from indoor dust collected in homes and, from debris isolated from wooden electricity poles (VGII and VNI) in the town of Santa Isabel do Rio Negro, located in deforested areas of the Amazon rainforest at the Rio Negro banks. The types of houses and the wooden poles of the city, with a predominance of wooden structures, are related to the generation of suitable substrates for recombination and expansion of the agents of cryptococcosis. Inhabitants should be informed about the risks associated with the use of untreated wood as construction material. Decomposed wood in and around the homes should be replaced with new pre-treated ones, as a simple measure that can prevent the spread of this fungus in the environment and future outbreaks in the region.

 

image description

Figure 2. Dust indoor sample DW-650 after 48 h incubation at 25 ºC plated on bird-seed agar, which resulted in uncountable dark brown colonies (suspected colonies of Cryptococcus spp.).

 

References:

  1. Trilles L, Lazéra Mdos S, Wanke B, Oliveira RV, Barbosa GG, Nishikawa MM, Morales BP, Meyer W. 2008. Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil. Mem Inst Oswaldo Cruz. 5:455-462.
  2. Brito Santos-F, Barbosa GG, Trilles L, Nishikawa MM, Wanke B, Meyer W, Carvalho-Costa FA, Lazera MS. 2015. Environmental isolation of Cryptococcus gattii VGII from indoor dust associate to typical wooden houses in the deep Amazon of the Rio Negro basin PLoS One.Feb 17;10(2): e0115866.
  3. Fortes ST, Lazéra MS, Nishikawa MM, Macêdo RCL, Wanke B. 2001.First isolation of Cryptococcus neoformans var. gattii from a native jungle tree in the Brazilian Amazon rainforest. Mycosis 44:137-140.
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  5. Engelthaler DM, Hicks ND, Gillece JD, Roe CC, Schupp JM, Driebe EM, Gilgado F, Carriconde F, Trilles L, Firacative C, Ngamskulrungroj P, Castañeda E, dos Santos Lazera M, Melhem MSC, Pérez-Bercoff Å, Huttlley G, Sorrell TC, Voelz K, May RC, Fisher MC, Thompson GR, III, Lockhart SR, Keim P, Meyer W. 2014. Cryptococcus gattii in North American Pacific Northwest: whole-population genome analysis provides insights into species evolution and dispersal. mBio, 5(4):e01464-14.

 

Acknowledgments

We are grateful for the contribution of the Postgraduate Program in Tropical Medicine, Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Brazil, and to students Souza AC, Barbosa ATL, Cabral AMA, Hardoim DJ, Camara DCP, Assunção LC, Silva MH, Portilho MM, Marques VA for their help in conducting the fieldwork. The authors acknowledge the Technological Platform Network of Oswaldo Cruz Foundation (RPT01A)/FIOCRUZ, Rio de Janeiro, Brazil. The work was supported by the “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES), Brazil, grant # 098/2012 to BW.

 

 

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