Consensus Multilocus Sequence Typing Scheme for Pneumocystis jirovecii

Abstract

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.

Keywords: CYB; P. jirovecii; consensus MLST scheme; mt26S and SOD; β-TUB.

Figure 1

Average amplification and sequencing rates for all targeted genetic loci. Calculations are based on the combined mean amplification and sequencing rates of both cohorts and are expressed as percentages. Error bars indicate the standard deviation.

Figure 2

Number of allele types identified amongst all studied isolates.

Figure 3

Phylogenetic tree of six P. jirovecii isolates (patients from Table 3) used in the case study to show the discriminatory power of the new consensus P. jirovecii MLST scheme, obtained by maximum likelihood analysis with the general time reversible (GTR) model with RaxML (version 7.2.8) using RaxmlGUI 1.1 [91], part of the software package MEGA ver. 7.0 [92].