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.

Genetic Polymorphisms of Pneumocystis jirovecii in HIV-Positive and HIV-Negative Patients in Northern China

“…The primers used in this study are listed in Table I . The PCR amplification conditions for β-Tub and 26S were the same as those previously reported ( Pasic et al 2020 ), and the conditions for other genes were the same as described in previous studies ( Lee et al 1998 ; Wang et al 2019 ; Xue et al 2019 ). DNA from P. jirovecii -positive specimens stored in our laboratory was used as the positive control.…”

“… ITS – internal transcribed spacer regions of rRNA operon, sod – superoxide dismutase, dhfr – dihydrofolate reductase, dhp – dihydropteroate synthase, WT – wild-type, cyb – cytochrome b, mt – mitochondrial large rRNA subunit, β-Tub – β-tubulin, 26S rRNA – 26S ribosomal RNA gene a – the genotype nomenclature based on previously published studies and b – the genotype locations according to the studies previously reported ( Walker et al 1998 ; Ma et al 1999 ; Beard et al 2000 ; Denis et al 2000 ; Takahashi et al 2002 ; Esteves et al 2010b ; Maitte et al 2013 ; Xue et al 2019 ; Pasic et al 2020 ) …”

“…Genetic polymorphisms of P. jirovecii in China 1 3 (Pasic et al 2020), and the conditions for other genes were the same as described in previous studies (Lee et al 1998;Wang et al 2019;Xue et al 2019). DNA from P. jirovecii-positive specimens stored in our laboratory was used as the positive control.…”

Tackling the emerging threat of antifungal resistance to human health

“…Unculturable fungi present a challenge, and more targeted methods are needed. For instance, a successful consensus multilocus sequence typing scheme for P. jirovecii 75 enables antifungal resistance marker analysis 76 . For Aspergillus spp., more knowledge of resistance mechanisms is required as many resistant isolates do not carry the few known resistance-associated alleles 77 .…”

Emerging Fungi and Diagnosis of Fungal Infections: Current Knowledge and New Developments

“…The evolution of molecular tools for the detection of fungal pathogens has been slow in the last decades, but has significantly advanced in recent years. Several of the reviews and articles deal with the diagnostic repertoire of the mycology laboratory and show the value of new and well-established tests [ 12 , 13 ].…”