Single nucleotide polymorphisms in <i>E. gingivalis</i> isolates.

<p>Single nucleotide polymorphisms in <i>E. gingivalis</i> isolates.</p

LSSP-PCR. Polyacrylamide gel (7.5%) stained with silver nitrate.

<p>Primers: A) EGO-1 and B) EGO-2. Lane 1: molecular marker 1 Kb (Invitrogen); lanes 2–25: HIV(+)/AIDS samples; lane 26: negative control.</p

Analysis of the genetic variability of the small ribosomal subunit in the 18S rRNA of <i>E. gingivalis</i> in clinical samples from HIV(+)/AIDS patients, detected by LSSP-PCR.

<p>M = male; F = female; CD4 =  CD4⁺ cell count; HIV− = HIV negative; UR = unrealized.</p

Phylogenetic relationship of <i>Entamoeba</i> spp. from HIV infected patients.

<p>Neighbor-joining method was performed in 24 taxa at the SSU rRNA locus. The optimal tree with the sum of branch length = 1.32 is shown and the bootstrap values were added to phylogenetic branches.</p

MLST-Based Population Genetic Analysis in a Global Context Reveals Clonality amongst Cryptococcus neoformans var. grubii VNI Isolates from HIV Patients in Southeastern Brazil

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

MLST-Based Population Genetic Analysis in a Global Context Reveals Clonality amongst <i>Cryptococcus neoformans</i> var. <i>grubii</i> VNI Isolates from HIV Patients in Southeastern Brazil

<div><p>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 <i>Cryptococcus neoformans</i> 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 <i>C</i>. <i>neoformans</i> var. <i>grubii</i> 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 <i>STE20</i>, <i>GPA1</i>, and <i>PAK1</i> 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 <i>C</i>. <i>neoformans</i> var. <i>grubii</i> VNI were identified that diverged from VNB around 0.58 to 4.8 million years ago.</p></div

Representative flow cytometry curves of selected southeastern Brazilian <i>Cryptococcus neoformans</i> var. <i>grubii</i> VNI isolates.

<p>Out of 143 isolates, 12 were identified as diploid. The haploid strains H99 <i>C</i>. <i>neoformans</i> var. <i>grubii</i> VNI and CDCR265 <i>C</i>. <i>gattii</i> VGII and the diploid hybrid strain WM 09.184 <i>C</i>. <i>neoformans</i> VNII/VNIV were included in each flow run as controls.</p

Multilocus linkage disequilibrium and recombination analyses amongst <i>Cryptococcus neoformans</i> var. <i>grubii</i> VNI continental populations.

<p>Multilocus linkage disequilibrium and recombination analyses amongst <i>Cryptococcus neoformans</i> var. <i>grubii</i> VNI continental populations.</p

Characteristics associated with deaths of the Brazilian patients.

<p>Characteristics associated with deaths of the Brazilian patients.</p

Coalescence gene genealogy of the <i>Cryptococcus neoformans</i> var. <i>grubii</i> VNI isolates evidencing the presence of two main clusters that separated 0.29 to 2.8 million years ago.

<p>Blue bars represent the 95% high posterior probability of the ages in the branches with a posterior probability limit of 0.5. The Bayesian posterior support values higher than 70% are described in the branches of the tree. One ST representing the VNB (ST7) and one representing the VNII (ST40) lineage were included in the analysis as out-groups. The continents of origin of the isolates are abbreviated as follows: AS: Asia, AF: Africa, EU: Europe, SA: South America, NA: North America.</p