Environmental Mapping of Paracoccidioides spp. in Brazil Reveals New Clues into Genetic Diversity, Biogeography and Wild Host Association

Abstract

Background: Paracoccidioides brasiliensis and Paracoccidioides lutzii are the etiological agents of Paracoccidioidomycosis (PCM), and are easily isolated from human patients. However, due to human migration and a long latency period, clinical isolates do not reflect the spatial distribution of these pathogens. Molecular detection of P. brasiliensis and P. lutzii from soil, as well as their isolation from wild animals such as armadillos, are important for monitoring their environmental and geographical distribution. This study aimed to detect and, for the first time, evaluate the genetic diversity of P. brasiliensis and P. lutzii for Paracoccidioidomycosis in endemic and non-endemic areas of the environment, by using Nested PCR and in situ hybridization techniques.

Methods/principal findings: Aerosol (n = 16) and soil (n = 34) samples from armadillo burrows, as well as armadillos (n = 7) were collected in endemic and non-endemic areas of PCM in the Southeastern, Midwestern and Northern regions of Brazil. Both P. brasiliensis and P. lutzii were detected in soil (67.5%) and aerosols (81%) by PCR of Internal Transcribed Spacer (ITS) region (60%), and also by in situ hybridization (83%). Fungal isolation from armadillo tissues was not possible. Sequences from both species of P. brasiliensis and P. lutzii were detected in all regions. In addition, we identified genetic Paracoccidioides variants in soil and aerosol samples which have never been reported before in clinical or armadillo samples, suggesting greater genetic variability in the environment than in vertebrate hosts.

Conclusions/significance: Data may reflect the actual occurrence of Paracoccidioides species in their saprobic habitat, despite their absence/non-detection in seven armadillos evaluated in regions with high prevalence of PCM infection by P. lutzii. These results may indicate a possible ecological difference between P. brasiliensis and P. lutzii concerning their wild hosts.

Fig 1. Collection areas of environmental samples (soil, aerosol and animals) in Brazil.

The evaluated states (RO, MG and GO) are highlighted in different colors and the municipalities of sampling in different traces indicated with arrows.

Fig 2. The ITS region with the location of oligo probes designed for the detection and differentiation of P. brasiliensis and P. lutzii, labeled with HRP and Texas Red, respectively.

Fig 3

A) Molecular Phylogenetic Analysis by of ITS locus revealed by Maximum Likelihood and Neighbor Joining methods, using the Jukes-Cantor model parameters with range correction. Replication percentages on the tree are grouped in the bootstrap test (1000 replicates) and shown next to the branches. The sequences related to environmental samples are identified by acronyms SO_GO (Soil of Goiás) and AR_GO (Aerosol Goiás), AR_MG (Aerosol Minas Gerais) and SO_RO (Soil of Rondônia). B) Median-joining network showing the unique haplotypes of the Soil Clades I and II. Circles are proportional to haplotype frequency and numbers of mutations are represented by black dots. Red circles represent hypothetical missing intermediates (median vectors).

Fig 4. Fungal structures (400X) visualized by FISH and TSA-FISH techniques for aerosol samples and controls.

A: aerosol samples from Goiás with DAPI. B: aerosol samples from Goiás with P. brasiliensis probe. C: aerosol sample from Rondônia, with DAPI. D: aerosol samples from Rondônia with P. brasiliensis probes. E: aerosol sample from Goiás with DAPI. F: aerosol sample from Goiás with P. lutzii probe. G and I: Histoplasma capsulatum with DAPI. H: Histoplasma capsulatum with P. brasiliensis probe (specificity control). J: Histoplasma capsulatum with P. lutzii probe (specificity control). K: isolate Pb01 (P. lutzii) with P. lutzii probe (positive control). L: isolate T16B1 (P. brasiliensis) with P. brasiliensis probe. The probe for P. brasiliensis is conjugated with Horseradish Peroxidase/HRP and P. lutzii probe is labeled with Texas Red/TXR, and genetic material is labeled with DAPI.

Fig 5. Brazilian sites and biomes where environmental detection (from soil, aerosol and armadillos) of Paracoccidioides spp. has been carried out.

The collection areas encompass the states of Minas Gerais (MG), Goiás (GO) and Rondônia (RO) states, to São Paulo (SP) and Pará (PA). Circles outside of the map indicate the percentage of positivity in each area for P. brasiliensis (green) and P. lutzii (red) by in situ hybridization for aerosol samples. The circles outside of the map in black and grey indicate the percentage of positivity in each area for P. brasiliensis and P. lutzii by Nested PCR for soil and aerosol samples. The white circle indicates the negative detection in the evaluated areas (N.D. = Not detected). The armadillos indicate the number of collected animals and the positivity for isolation of Paracoccidioides brasiliensis (Pb) in each location of our study (in RO, GO and MG) and from previous works in SP [16] and PA [33]. The triangles (green), squares (yellow) and circles (red) show the distribution of the clinical isolates for cryptic species (S1, PS2 and P. lutzii) in previous studies [14,31].