Genetic investigations of pneumocystis jirovecii : detection, cotrimoxazole resistance and population structure
Date
2005-12
Authors
Robberts, Frans Jacob Lourens
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : University of Stellenbosch
Abstract
Pneumocystis jirovecii is a significant contributor to the burden of disease in
immunocompromised patients. The polymerase chain reaction (PCR) is more
sensitive and specific than microscopy. Cotrimoxazole prophylactic breakthrough and
treatment failures have been reported, and associated with mutations at codons 55
and 57 of P. jirovecii dihydropteroate synthase (DHPS). No phylogenetic or
population genetic models have been successful in elucidating P. jirovecii
intraspecies strain relatedness.
Aims: 1) Compare detection rates of nine PCR techniques and immunofluorescence
microscopy (IF); 2) Determine the extent of co-infecting pathogens associated with
Pneumocystis Pneumonia (PcP); 3) Determine local P. jirovecii ITS1-5.8S-ITS2 rDNA
strain types, and model lineage evolution employing a coalescent-theory based
statistical parsimony network analysis; 4) Investigate the possible emergence of
cotrimoxazole-resistant strains
Methods: PCR was evaluated on clinical specimens employing: ITS nested; DHPS
single and nested; DHFR nested; major surface glycoprotein (MSG) heminested;
mitochondrial large subunit rRNA (mtLSUrRNA) single and nested; 18S rRNA onetube
nested, and real-time 5S rRNA PCR. Retrospective analysis of co-infecting
pathogens seen in PcP patients was conducted. ITS regions were amplified, cloned
and sequenced. Statistical parsimony was applied for coalescence based network
genotype analysis. DHPS genome walking was attempted and DHPS and DHFR
primer annealing sites explored. Amplified DHPS and DHFR genes were cloned and
sequenced.
Results: Most sensitive PCR technique was mtLSUrRNA nested followed by 5S realtime
PCR. A poor correlation exist between mtLSUrRNA PCR and IF. Review of
clinical records suggested a high rate of false-positive IF results. P. jirovecii was
detected in 4.3% M. tuberculosis-positive HIV-positive, and 2.5% M. tuberculosispositive
HIV-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 was
detected in three patients and designated u. More than one strain type was detected
in 15/19 patients. Analysis of 5.8SrDNA region revealed 13 clones containing 1-2
nucleotide polymorphisms. Of 85 mtLSUrRNA PCR-positive specimens, currently
employed primers amplified DHPS and DHFR genes from 53 and 27 specimens,
respectively. Newly designed DHPS primers increased detection in 3 / 28 previously
DHPS-negative mtLSUrRNA-positive specimens. Of 56 DHPS genes amplified and
sequenced, one contained the double mutation (Thr55Aa; Pro57Ser). DHFR
Ala67Val 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 local
strains. It is recommended that mtLSUrRNA PCR be performed in parallel to IF and
discordant results resolved with clinical evaluation. Co-infection with P. jirovecii and
M. tuberculosis occurs in South Africa, and treatment for both pathogens is
recommended when demonstrated by the laboratory. ITS genotyping employing
statistical parsimony network analysis suggests type Eg as major ancestral
haplotype, and supports recombination contributing to strain diversity worldwide.
DHPS mutations may signal emergence of resistance to cotrimoxazole in South
Africa, however, low sensitivity of primers limits surveillance efforts.
Description
Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2005.
Keywords
Polymerase chain reaction, Pneumocystis carinii pneumonia, Tuberculosis, Drug resistance, Dissertations -- Medical microbiology, Theses -- Medical microbiology