6/6 markers in samples taken between 0 and 14 days apart and in all

6/6 markers in samples taken between 0 and 14 days apart and in all 15 patients, in 5/6 markers in samples taken between 0 and 37 days apart. The analysis revealed the occurrence of allele replacement or modification (contraction or extension of 1 to 6 repeats for a given marker at 1 or 2 loci) for 4/15 patients, suggesting the microevolution of P. jirovecii during infection (over a short period of time, up to 37 days). This reveals rapid variation of these parts of the genome due to their repetitive nature. Indeed, infection is associated with fungal proliferation and modification, and the number of repeat units could be easily altered (gain or loss). Unfortunately, the stability of each selected marker, as was reported for STRAf in Aspergillus fumigatus [31], cannot bePLOS ONE | DOI:10.1371/journal.pone.0125763 May 1,12 /STR-Typing for P. jiroveciiTable 5. Discriminatory power of the assay for genotyping P. jirovecii. Name of markers included No. of markers included Whole data (n = 54 patients) STRPj_138/189/279 STRPj_022/138/189/279 STRPj_022/138/189/278/ 279 STRPj_022/108/138/189/ 278/279 3 4 5 6 0.949 0.963 0.980 0.985 Simpson’s index of diversity (D) wo cluster 2 Gt (n = 45 patients) 0.957 0.981 0.989 0.992 wo renal transplant of cluster 2 (n = 45 patients) 0.964 0.983 0.990 0.doi:10.1371/journal.pone.0125763.tassessed experimentally in humans during infection nor in vitro with long-term cultures of P. jirovecii. Trichostatin AMedChemExpress TSA However, the culture system published recently [24] or murine model of Pneumocystis pneumonia with P. murina could be used to test the stability of the markers and to study the plasticity of P. jirovecii nuclear and mitochondrial genomes. Comparison of STR to sequencebased typing methods will also bring new arguments to assess the level of stability of these STR markers. In our study, about 70 samples harbored multiple genotypes. Sanger sequencing-based methods are less sensitive and detected multiple genotypes in only about 30 of samples [28,47]. A high number of multiple genotypes were also observed with SSCP studies in HIV patients [48] or with an STR-based method (70 of patients) [44], suggesting that different strains of P. jirovecii are constantly transmitted between humans throughout life [1,2]. Recovery of unique versus multiple genotypes was not significantly associated with underlying disease, although samples from renal transplant patients tended to harbor a unique genotype, thus reinforcing the hypothesis of transmission between these patients, as already described in literature [18,19,22]. The ability to detect multiple genotypes makes the data ACY-241 supplier difficult to interpret but enables the identification of minor alleles. Association of major and minor allele has been used to determine genotypes using microsatellite [44] and SSCP [46] typing methods. From a technical point of view, we think that PCR amplification of STR markers could introduce variation in the allele ratio in mixed samples. Indeed, a variation (2.3 to 9.4 ) was observed in the proportion of the minority allele compared to the major allele. Consequently, it is difficult to be confident in the results when determining the different genotypes in complex allele mixtures (multiple alleles at more than one marker) including various minority alleles, unless one knows precisely what to look for, as was the case for Gt21. We searched for each of the 61 genotypes determined in pure or mixed samples at one locus in mixed samples two or three alleles at two.6/6 markers in samples taken between 0 and 14 days apart and in all 15 patients, in 5/6 markers in samples taken between 0 and 37 days apart. The analysis revealed the occurrence of allele replacement or modification (contraction or extension of 1 to 6 repeats for a given marker at 1 or 2 loci) for 4/15 patients, suggesting the microevolution of P. jirovecii during infection (over a short period of time, up to 37 days). This reveals rapid variation of these parts of the genome due to their repetitive nature. Indeed, infection is associated with fungal proliferation and modification, and the number of repeat units could be easily altered (gain or loss). Unfortunately, the stability of each selected marker, as was reported for STRAf in Aspergillus fumigatus [31], cannot bePLOS ONE | DOI:10.1371/journal.pone.0125763 May 1,12 /STR-Typing for P. jiroveciiTable 5. Discriminatory power of the assay for genotyping P. jirovecii. Name of markers included No. of markers included Whole data (n = 54 patients) STRPj_138/189/279 STRPj_022/138/189/279 STRPj_022/138/189/278/ 279 STRPj_022/108/138/189/ 278/279 3 4 5 6 0.949 0.963 0.980 0.985 Simpson’s index of diversity (D) wo cluster 2 Gt (n = 45 patients) 0.957 0.981 0.989 0.992 wo renal transplant of cluster 2 (n = 45 patients) 0.964 0.983 0.990 0.doi:10.1371/journal.pone.0125763.tassessed experimentally in humans during infection nor in vitro with long-term cultures of P. jirovecii. However, the culture system published recently [24] or murine model of Pneumocystis pneumonia with P. murina could be used to test the stability of the markers and to study the plasticity of P. jirovecii nuclear and mitochondrial genomes. Comparison of STR to sequencebased typing methods will also bring new arguments to assess the level of stability of these STR markers. In our study, about 70 samples harbored multiple genotypes. Sanger sequencing-based methods are less sensitive and detected multiple genotypes in only about 30 of samples [28,47]. A high number of multiple genotypes were also observed with SSCP studies in HIV patients [48] or with an STR-based method (70 of patients) [44], suggesting that different strains of P. jirovecii are constantly transmitted between humans throughout life [1,2]. Recovery of unique versus multiple genotypes was not significantly associated with underlying disease, although samples from renal transplant patients tended to harbor a unique genotype, thus reinforcing the hypothesis of transmission between these patients, as already described in literature [18,19,22]. The ability to detect multiple genotypes makes the data difficult to interpret but enables the identification of minor alleles. Association of major and minor allele has been used to determine genotypes using microsatellite [44] and SSCP [46] typing methods. From a technical point of view, we think that PCR amplification of STR markers could introduce variation in the allele ratio in mixed samples. Indeed, a variation (2.3 to 9.4 ) was observed in the proportion of the minority allele compared to the major allele. Consequently, it is difficult to be confident in the results when determining the different genotypes in complex allele mixtures (multiple alleles at more than one marker) including various minority alleles, unless one knows precisely what to look for, as was the case for Gt21. We searched for each of the 61 genotypes determined in pure or mixed samples at one locus in mixed samples two or three alleles at two.