Genotypic analysis of clinical and environmental Cryptococcus neoformans isolates from Brazil reveals the presence of VNB isolates and a correlation with biological factors

Leonardo Euripedes Andrade-Silva,Natalia Evelyn Araujo,Helioswilton Sales-Campos,Wieland Meyer,Fernanda Machado Fonseca,Anderson Vilas-Boas,Juliana Andrade-Silva,Kennio Ferreira-Paim,Marcus Vinicius Da Silva,Bruno Da Silva Prudente,Thatiana Bragine Ferreira,Verônica Morais Manzato,Mario Léon Silva-Vergara,Virmondes Rodrigues Júnior,Kelli Buosi,Delio José Mora,Kirsten Nielsen

doi:10.1371/journal.pone.0193237

Abstract

Cryptococcal infections are mainly caused by members of the Cryptococcus neoformans species complex (molecular types VNI, VNII, VNB, VNIV and the AD hybrid VNIII). PCR of the mating type loci and MLST typing using the ISHAM-MLST consensus scheme were used to evaluate the genetic relationship of 102 (63 clinical and 39 environmental) C. neoformans isolates from Uberaba, Brazil and to correlate the obtained genotypes with clinical, antifungal susceptibility and virulence factor data. All isolates were mating type alpha. MLST identified 12 known and five new sequence types (ST). Fourteen STs were identified within the VNI isolates, with ST93 (57/102, 56%) and ST77 (19/102, 19%) being the most prevalent. From the nine VNII isolates previously identify by URA5-RFLP only four (ST40) were confirmed by MLST. The remaining five grouped within the VNB clade in the phylogenetic analysis corresponding to the sequence type ST504. Other two environmental isolates also grouped within VNB clade with the new sequence type ST527. The four VNII/ST40 isolates were isolated from CSF. The two VNIV sequence types (ST11 and ST160) were isolated from blood cultures. Two of six patients evaluated with more than one isolates had mixed infections. Amongst the VNI isolates 4 populations were identified, which showed differences in their susceptibility profiles, clinical outcome and virulence factors. These results reinforce that ST93 is the most prevalent ST in HIV-infected patients in the Southeastern region of Brazil. The finding of the VNB molecular type amongst environmental Brazilian isolates highlights that this genotype is not restricted to the African continent.

Highlights

Cryptococcosis causes high mortality in HIV/AIDS patients in limited-resource settings, mainly in sub-Saharan Africa, with a 70% mortality rate being described [1]
A high variability of isolates from some geographical areas from the African continent compared to other regions highlighted the relevance to characterize isolates of C. neoformans from different sources and geographical locations in order to outline the global distribution of this species [11,12,13]
Underlying clinical information was available for 59 patients, the majority of them (57) had AIDS, and two were kidney transplant recipients (S1 and S2 Tables)

Summary

Introduction

Cryptococcosis causes high mortality in HIV/AIDS patients in limited-resource settings, mainly in sub-Saharan Africa, with a 70% mortality rate being described [1]. VNI and VNII are worldwide distributed, VNB appears to be endemic to Africa [6], but some reports have suggested a more global distribution [7,8], and VNIV seems to be endemic in Europe [9] The identification of these molecular types is usually based on the widely used MLST sequencing protocol standardized by the International Society for Human and Animal Mycology (ISHAM). MLST permits to establish better comparisons of the results obtained around the world and favors a evolutionary relationship analysis [10] Using this scheme, a high variability of isolates from some geographical areas from the African continent compared to other regions highlighted the relevance to characterize isolates of C. neoformans from different sources and geographical locations in order to outline the global distribution of this species [11,12,13]

Methods

Results

Conclusion