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    • aromatase inhibitor The following are the supplementary data

    2018-11-03

    The following are the supplementary data related to this article.
    Funding Sources This work was supported by the National Mega-projects of Science Research for the 12th Five-year Plan of China grant no. 2012ZX10003008-005, the National High Technology Research and Development of China grant no. 2012AA02A401, Wuhan Bureau of Science and Technology grant no. 2015060101010029, and the Fundamental Research Funds for the Central Universities grants HUST no. 2015MS098 and no. 2015ZZGH012. The funders had no role in study design, data collection, data analysis, interpretation, and writing of the report.
    Conflicts of Interest
    Patent Application
    Author Contributions
    Acknowledgements
    Introduction Pneumocystis jirovecii is a life-threatening atypical fungal organism for which our knowledge of its epidemiology and basic biology is limited, largely due to an inability to reproducibly culture the microorganism in vitro (Thomas and Limper, 2007). Pneumocystis species has been coevolving with each mammal species for 100 millions years leading to the specificity of P. jirovecii to humans (Aliouat-Denis et al., 2008). Individuals are exposed to P. jirovecii in childhood, with >80% of children being seropositive before the age of 2years (Bishop and Kovacs, 2003; Meuwissen et al., 1977; Peglow et al., 1990; Pifer et al., 1978; Vargas et al., 2001). This organism is therefore considered as a pulmonary resident of immunocompetent individuals who carry P. jirovecii, at least transiently, in their respiratory tract (Gigliotti and Wright, 2012). P. jirovecii can then circulate constantly between normal hosts as suggested by the high proportion of mixed infections (Alanio and Bretagne, 2017). Genotyping systems have been developed in the last 20years to explore the pathophysiology of the infection (Pneumocystis pneumonia, PCP) and to trace the transmission of P. jirovecii in specific areas such as hospital wards. PCR single strand conformation polymorphism (PCR-SSCP) of nuclear and mitochondrial loci allowed the analysis of several isolates associated with clustered infections (Hauser et al., 1997a,b; Nahimana et al., 2000). Sequencing of internal transcribed spacers (ITS), which locus is unique in the nuclear aromatase inhibitor (Helweg-Larsen et al., 2001; Le Gal et al., 2012; Tsolaki et al., 1996), and sequencing of mitochondrial large subunit ribosomal loci have also been used (Keely et al., 1995). A multi-locus sequence typing (MLST) approach has also been implemented (Keely and Stringer, 1996) and a MLST scheme was optimized based on the sequencing of eight nuclear and mitochondrial loci (Maitte et al., 2013). The number of different genotypes described in literature was 43 using the PCR-SSCP method (Hauser, 2004), although >60 genotypes were identified using ITS sequencing methods (Lu and Lee, 2008). Single nucleotide polymorphism (SNP) was also studied using single nucleotide primer extension in mitochondrial and genomic genes (Alanio et al., 2015; Esteves et al., 2011) and more recently, using ultra-deep-pyrosequencing (UDPS) to study three mitochondrial SNPs (Alanio et al., 2016). An important feature of genotyping methods is the sensitivity when detecting minority alleles, since for P. jirovecii genotyping is performed directly on clinical samples because of the absence of culture. Although mixtures of two or more genotypes in one clinical sample have been reported, the rate of mixed infection varied from 5 to 25% using classical Sanger DNA sequencing (Esteves et al., 2010, 2008; Helweg-Larsen et al., 2001) and single nucleotide primer extension, respectively (Alanio et al., 2015; Esteves et al., 2011), to 70% by both PCR-SSCP (Hauser et al., 2001) and Microsatellite Length Polymorphism (MLP) (Gits-Muselli et al., 2015; Parobek et al., 2014), and to >90% of the samples using UDPS (Alanio et al., 2016). To avoid sequencing and to test genetic variation differently to SNPs, MLP schemes have also been recently developed, targeting Short Tandem Repeat (STR) markers (Gits-Muselli et al., 2015; Parobek et al., 2014) identified once the P. jirovecii genome was available (Cissé et al., 2012). The first MLP genotyping method developed by Parobek et al., based on eight genetically unlinked markers, was used to genotype 63 clinical isolates from Africa (Uganda), Europe (Spain), and USA (San Francisco) (Parobek et al., 2014). A different MLP genotyping method based on six other STR markers was more recently developed (Gits-Muselli et al., 2015). This last MLP typing method is reproducible, cheap, with a high throughput and a high discriminatory power (DP) of 0.992 making it interesting to investigate transmission in hospital settings (Gits-Muselli et al., 2015; Robin et al., 2017).