Description CryHs Study

Rationale of the study

Multi-locus sequence typing (MLST) is the gold standard for genotyping Cryptococcus isolates, however, the standard primers have been designed to amplify and sequence haploid isolates, therefore diploid/aneuploid hybrids cannot be genotyped. This is an important issue considering that in Europe the cases of cryptococcosis due to hybrids within C. neoformans species complex represent more than 20% out of the total cases, whereas in the other continents prevalence can range from 1 to 15%. Recently, the Mycology Laboratory at Università degli Studi di Milano, Italy, in collaboration with the University of Montpellier, France, have designed new allele-specific primers to amplify and sequence the seven loci of the standard ISHAM MLST scheme (CAP59, IGS1, GPD1, LAC1, PLB1, SOD1, URA5) in C. neoformans species complex hybrids (Cogliati et al. 2020). This provides a new tool to genotype AD hybrids allowing phylogenetic and population genetic studies in comparison with haploid populations.

Aim of the study

To sequence and genotype a large number of AD hybrid strains from Europe and other continents using the new MLST primers.

To compare AD-hybrid genotypes to haploid genotypes present in the global MLST database (www.mycologylab.org).

To elucidate the phylogenetic position of AD hybrids and their role in the evolution of Cryptococcus.

How to fulfill the goal

The strategy to reach the goal in a short time is to obtain a large participation to the study including several research centers able to sequence Cryptococcus isolates.

We expect the participation of about 10 research centers and more than 100 isolates sequenced.

Isolates

The AD-hybrid isolates to be sequenced in each center could be isolates present in the culture collection of each center or isolates sent by the coordinator center.

A list of all isolates that will be sequenced will be published in the Screen Project website.

Primers and PCR conditions

Primers and PCR conditions can be found at the following link.

Results

All sequence results will be sent to the coordinator center (Lab. Micologia Medica, Università degli Studi di Milano, Italy, massimo.cogliati@unimi.it).

Then sequences will be deposited in the global MLST database (www.mycologylab.org).

Duration of the study

The study will go on until all sequencing are completed. We expect to obtain all results within the end of 2021.