Pneumocystis jirovecii is an opportunistic human pathogenic fungus causing severe pneumonia mainly in immunocompromised hosts. Multilocus sequence typing (MLST) remains the gold standard for genotyping of this unculturable fungus. However, the lack of a consensus scheme impedes a global comparison, large scale population studies and the development of a global MLST database. To overcome this problem this study compared all genetic regions (19 loci) currently used in 31 different published Pneumocystis MLST schemes. The most diverse/commonly used eight loci, β-TUB, CYB, DHPS, ITS1, ITS1/2, mt26S and SOD, were further assess for their ability to be successfully amplified and sequenced, and for their discriminatory power. The most successful loci were tested to identify genetically related and unrelated cases. A new consensus MLST scheme consisting of four genetically independent loci: β-TUB, CYB, mt26S and SOD, is herein proposed for standardised P. jirovecii typing, successfully amplifying low and high fungal burden specimens, showing adequate discriminatory power, and correctly identifying suspected related and unrelated isolates. The new consensus MLST scheme, if accepted, will for the first time provide a powerful tool to investigate outbreak settings and undertake global epidemiological studies shedding light on the spread of this important human fungal pathogen.

Cryptococcosis caused by yeasts of the Cryptococcus gattii species complex is an increasingly important mycological disease in humans and other mammals. In Australia, cases of C. gattii-related cryptococcosis are more prevalent in the koala (Phascolarctos cinereus) compared to humans and other animals, likely due to the close association that both C. gattii and koalas have with Eucalyptus species. This provides a cogent opportunity to investigate the epidemiology of spontaneous C. gattii infections in a free-living mammalian host, thereby offering insights into similar infections in humans. This study aimed to establish a link between nasal colonisation by C. gattii in free-ranging koalas and the tree hollows of Eucalyptus species, the key environmental source of the pathogen. We (i) detected and genotyped C. gattii from nine out of 169 free-ranging koalas and representative tree hollows within their home range in the Liverpool Plains, New South Wales, and (ii) examined potential environmental predictors of nasal colonisation in koalas and the presence of C. gattii in tree hollows. Phylogenetic analyses based on multi-locus sequence typing (MLST) revealed that the koalas were most likely colonised by the most abundant C. gattii genotypes found in the Eucalyptus species, or closely related genotypes. Importantly, the likelihood of the presence of C. gattii in tree hollows was correlated with increasing hollow size.

Pneumocystis jirovecii is associated with non-noxious colonization or severe pneumonia in immunocompromised hosts. Epidemiological investigations have been hampered by the lack of a standardized typing scheme. Thus, only partial molecular data on Spanish P. jirovecii cases are available. Recently, a new ISHAM consensus multilocus sequence typing scheme (MLST) targeting β-TUB, mt26S, CYB, and SOD with a publicly accessible database has been launched to overcome this problem. The molecular epidemiology of P. jirovecii from immunocompromised patients either colonized (n = 50) or having pneumonia (n = 36) seen between 2014 and 2018 at a single center in Barcelona, Spain, was studied. The new ISHAM consensus MSLT scheme was used to investigate the local epidemiology and identify possible unnoticed outbreaks. Mutations in the DHPS gene, not included in the scheme but giving information about potential sulfa treatment failure, were also studied. The study assigned 32 sequence types (ST) to 72.2% pneumonia and 56% colonization cases. The most frequent STs were ST21 (18.5%), ST22 (14.8%), and ST37(14.8%). For non-unique STs, ST3, ST30 and ST31 were found only in pneumonia cases, whereas ST27 was associated exclusively to colonizations. Despite 38 patients sharing similar STs, only two were involved in a potential cross transmission event. No DHPS mutations were identified. The new consensus typing scheme was useful to ascertain the molecular epidemiology of P. jirovecii in our center revealing a high genetic diversity and the potential association of specific STs to colonization and pneumonia cases. Lay Summary: A newly described MLST scheme aims at providing a standardized tool to study and compare Pneumocystis jirovecii epidemiology. A high diversity among P. jirovecii isolates from patients in Barcelona, Spain, and a potential association between specific STs and infection/colonization were identified.