Correlation Between Pneumocystis jirovecii Mitochondrial Genotypes and High and Low Fungal Loads Assessed by Single Nucleotide Primer Extension Assay and Quantitative Real‐Time PCR. (27th May 2015)

• Record Type: Journal Article
• Authors: Alanio, Alexandre; Olivi, Martine; Cabaret, Odile; Foulet, Françoise; Bellanger, Anne‐Pauline; Millon, Laurence; Berceanu, Ana; Cordonnier, Catherine; Costa, Jean‐Marc; Bretagne, Stéphane
• Is Part Of: Journal of eukaryotic microbiology. Volume 62:Number 5(2015:Sep./Oct.)
• Page Start: 650
• Page End: 656
• Publication Date: 2015-05-27
• Subjects: Protista -- Periodicals; Eukaryotic cells -- Periodicals; Microbiology -- Periodicals; 579.05
• DOI: 10.1111/jeu.12222
• Languages: English
• ISSNs: 1066-5234

Abstract

<abstract abstract-type="main" id="jeu12222-abs-0001"> <title>Abstract</title> <p>We designed a single nucleotide primer extension (SNaPshot) assay for <italic>Pneumocystis jirovecii</italic> genotyping, targeting <italic>mt85 </italic>SNP of the mitochondrial large subunit ribosomal RNA locus, to improve minority allele detection. We then analyzed 133 consecutive bronchoalveolar lavage (BAL) fluids tested positive for <italic>P. jirovecii </italic>DNA by quantitative real‐time PCR, obtained from two hospitals in different locations (Hospital 1 [<italic>n </italic>=<italic> </italic>95] and Hospital 2 [<italic>n </italic>=<italic> </italic>38]). We detected three different alleles, either singly (<italic>mt85C</italic>: 39.1%; <italic>mt85T</italic>: 24.1%; <italic>mt85A</italic>: 9.8%) or together (27%), and an association between <italic>P. jirovecii mt85</italic> genotype and the patient's place of hospitalization (<italic>p </italic>=<italic> </italic>0.011). The lowest fungal loads (median = 0.82 × 10<sup>3</sup> copies/μl; range: 15–11 × 10<sup>3</sup>) were associated with <italic>mt85</italic>A and the highest (median = 1.4 × 10<sup>6</sup> copies/μl; range: 17 × 10<sup>3</sup>–1.3 × 10<sup>7</sup>) with <italic>mt85CTA</italic> (<italic>p </italic>=<italic> </italic>0.010). The ratios of the various alleles differed between the 36 mixed‐genotype samples. In tests of serial BALs (median: 20 d; range 4–525) from six patients with mixed genotypes, allele ratio changes were observed five times and genotype replacement once. Therefore, allele ratio changes seem more frequent than genotype replacement when using a SNaPshot assay more sensitive for detecting minority alleles than Sanger sequencing. Moreover, because microscopy detects only high fungal loads, the selection of microscopy‐positive samples may miss genotypes associated with low loads.</p> </abstract>