Genotyping and Coalescent Phylogenetic Analysis of Pneumocystis jiroveci from South Africa

Abstract

Sequence analysis of Pneumocystis jiroveci internal transcribed spacer (ITS) regions has become an important epidemiological tool. The objectives of the present study were to investigate sequence variations in the ITS1-5.8S ribosomal DNA (rDNA)-ITS2 regions; determine the P. jiroveci genotypes present in Cape Town, South Africa; and resolve the lineage evolution of the types by use of the coalescent theory. ITS regions were amplified from samples collected from 19 patients. PCR products were cloned, and four to five clones were sequenced from each specimen. Statistical parsimony was applied for coalescence-based network genotype analysis. The most prevalent type was Eg (14 of 19 patients, 33 of 83 clones), followed by Gg (4 of 19 patients, 7 of 83 clones), Eu (3 of 19 patients, 5 of 83 clones), and Gh (2 of 19 patients, 2 of 83 clones). Four new combinations (Eo, Je, Ge, and No), 11 new ITS1 sequences, and 13 new ITS2 sequences were identified. A new ITS2 type was detected in three patients and was designated type u. Coinfection appeared to be common, with 15 of 19 patients harboring more than one type and with up to six types per specimen. The resultant parsimony network identified Eg as the most probable ancestral haplotype and supported the occurrence of recombinational events within the population studied. Although the 5.8S rDNA region revealed only 13 clones containing one to two nucleotide polymorphisms, it may assist in defining types. Coalescent theory proposed that Eg is an ancestral type from which microevolutionary subtypes radiate.Pneumocystis pneumonia (PCP) is a major contributor to morbidity and mortality in immunocompromised individuals (16,18). Many molecular epidemiological techniques are not applicable for the typing of Pneumocystis, as it cannot readily be propagated in vitro. Regions that have been investigated for use in the design of a typing method include the mitochondrial large-subunit rRNA, mitochondrial small-subunit rRNA, the arom locus, and internal transcribed spacer (ITS) regions (21). The sequence diversity of the Pneumocystis jiroveci ITS1 and ITS2 regions prompted these regions to be the basis on which typing of P. jiroveci could be conducted (10). Ribosomal DNA (rDNA) of P. jiroveci is present as a single copy in the cell and is transcribed as a single transcript, with 18S rRNA, 5.8S rRNA, and 26S rRNA occurring in tandem (4). The rRNA genes are separated by the ITS1 region between 18S rRNA and 5.8S rRNA and the ITS2 region between 5.8S rRNA and 26S rRNA (3, 6). Globally, the most frequently encountered genotypes are Eg and Ne (9, 20, 21). Latouche et al. (8) proposed that as a specific type was seen to persist during the same episode of PCP, genotype switching did not occur. However, in a study conducted with 19 patients during the same episode of PCP, genotype changes were observed in 53% of the patients (5) and coinfection with more than one genotype has been reported in a high proportion of PCP episodes (10,12,14,20,21,22). The present understanding is that P. jiroveci inf...

Frequent In Vitro Recombination in Internal Transcribed Spacers 1 and 2 during Genotyping of Pneumocystis jirovecii

“…In addition to these observations, ITS typing data have recently been presented as indicative of the occurrence of genetic recombination in P. jirovecii populations (17,21). Although the results reported here cannot be used to draw specific conclusions about clonal or recombining modes of reproduction in P. jirovecii, they do suggest that meiotic recombination in the rDNA of this fungus is not a frequent event.…”

“…To date, more than 30 ITS1 genotypes and 40 ITS2 genotypes with more than 90 haplotypes (combinations of ITS1 and ITS2 types) have been described worldwide, based on two different but similar typing systems (8,26). Some ITS haplotypes are globally more common, and coinfections with multiple types of P. jirovecii often occur (4,8,11,15,17,25,26). Although these typing systems have been useful, questions have been raised about the extensive polymorphism and stability of the ITS locus and its use for the genotyping of P. jirovecii.…”

Clinical Significance and Phylogenetic Relationship of Novel Australian Pneumocystis jirovecii Genotypes

“…P. jirovecii ITS genotypes were aligned with previously published and unpublished genotypes (GenBank accession no. AF013806 to AF013834, AF374238 to AF374265, AF498265, AF135711 to AF135712, AY328043 to AY328066, AY550105 to AY550109, and AF013835 to AF013840) (13,23,24,26,27,29,31). The mt LSU rRNA genotypes were characterized based on the polymorphic sites at codons 85 and 248: genotypes 1 (85:C/248:C), 2 (85:A/248:C), 3 (85:T/248:C), and 4 (85:C/248:T) (12).…”

Dihydropteroate Synthase and Novel Dihydrofolate Reductase Gene Mutations in Strains of Pneumocystis jirovecii from South Africa

“…Sequencing of ITS regions from five clones per specimen demonstrated that the P. jirovecii isolates harboring the DHFR C200T transitions had ITS genotypes Eg and Eu. Interestingly, ITS type Eu, which to date has only been found in South Africa (12), was associated with the DHFR mutation Ala67Val. Furthermore, the DHFR mutation Arg59Gly and the intron transition mutation C278T have not previously been described.…”

“…For strain typing, amplification of the internal transcribed spacer (ITS) region of the rRNA gene was performed (7), and PCR products were subcloned prior to sequencing (12). The ITS sequences obtained were compared to those of published ITS regions, and type nomenclature was assigned in accordance with Lee et al as described by Robberts et al (12).…”