Genetic investigations of pneumocystis jirovecii : detection, cotrimoxazole resistance and population structure

[摘要] Pneumocystis jirovecii is a significant contributor to the burden of disease inimmunocompromised patients. The polymerase chain reaction (PCR) is moresensitive and specific than microscopy. Cotrimoxazole prophylactic breakthrough andtreatment failures have been reported, and associated with mutations at codons 55and 57 of P. jirovecii dihydropteroate synthase (DHPS). No phylogenetic orpopulation genetic models have been successful in elucidating P. jiroveciiintraspecies strain relatedness.Aims: 1) Compare detection rates of nine PCR techniques and immunofluorescencemicroscopy (IF); 2) Determine the extent of co-infecting pathogens associated withPneumocystis Pneumonia (PcP); 3) Determine local P. jirovecii ITS1-5.8S-ITS2 rDNAstrain types, and model lineage evolution employing a coalescent-theory basedstatistical parsimony network analysis; 4) Investigate the possible emergence ofcotrimoxazole-resistant strainsMethods: PCR was evaluated on clinical specimens employing: ITS nested; DHPSsingle and nested; DHFR nested; major surface glycoprotein (MSG) heminested;mitochondrial large subunit rRNA (mtLSUrRNA) single and nested; 18S rRNA onetubenested, and real-time 5S rRNA PCR. Retrospective analysis of co-infectingpathogens seen in PcP patients was conducted. ITS regions were amplified, clonedand sequenced. Statistical parsimony was applied for coalescence based networkgenotype analysis. DHPS genome walking was attempted and DHPS and DHFRprimer annealing sites explored. Amplified DHPS and DHFR genes were cloned andsequenced.Results: Most sensitive PCR technique was mtLSUrRNA nested followed by 5S realtimePCR. A poor correlation exist between mtLSUrRNA PCR and IF. Review ofclinical records suggested a high rate of false-positive IF results. P. jirovecii wasdetected in 4.3% M. tuberculosis-positive HIV-positive, and 2.5% M. tuberculosispositiveHIV-negative patients. P. jirovecii was detected in 45% HIV-negative patients. The most prevalent ITS type was Eg. Four new combinations: Eo, Je, Ge,No; 11 new ITS1 and 13 new ITS2 sequences were identified. A new ITS2 type wasdetected in three patients and designated u. More than one strain type was detectedin 15/19 patients. Analysis of 5.8SrDNA region revealed 13 clones containing 1-2nucleotide polymorphisms. Of 85 mtLSUrRNA PCR-positive specimens, currentlyemployed primers amplified DHPS and DHFR genes from 53 and 27 specimens,respectively. Newly designed DHPS primers increased detection in 3 / 28 previouslyDHPS-negative mtLSUrRNA-positive specimens. Of 56 DHPS genes amplified andsequenced, one contained the double mutation (Thr55Aa; Pro57Ser). DHFRAla67Val was detected in three specimens and a new DHFR genotype (Arg59Gly;C278T) was demonstrated.Conclusions: The study emphasises the need to evaluate PCR primers against localstrains. It is recommended that mtLSUrRNA PCR be performed in parallel to IF anddiscordant results resolved with clinical evaluation. Co-infection with P. jirovecii andM. tuberculosis occurs in South Africa, and treatment for both pathogens isrecommended when demonstrated by the laboratory. ITS genotyping employingstatistical parsimony network analysis suggests type Eg as major ancestralhaplotype, and supports recombination contributing to strain diversity worldwide.DHPS mutations may signal emergence of resistance to cotrimoxazole in SouthAfrica, however, low sensitivity of primers limits surveillance efforts.

[发布机构] Stellenbosch University