Genotyping and phylogenetic analysis of Pneumocystis jirovecii isolates from India

Pneumocystis jirovecii is the cause of Pneumocystis pneumonia (PCP) in immuno-compromised individuals. The aim of this study was to describe the genotypes/haplotypes of P. jirovecii in immuno-compromised individuals with positive polymerase chain reaction (PCR) result for PCP. The typing was based on sequence polymorphism at internal transcribed spacer (ITS) regions of rRNA operon. Phylogenetic relationship between Indian and global haplotypes was also studied. Between January 2005 to October 2008, 43 patients were found to be positive for Pneumocystis using PCR targeting mitochondrial large subunit rRNA (mt LSU rRNA) and ITS region. Genotyping of all the positive samples was performed at the ITS locus by direct sequencing. Nine ITS1 alleles (all previously known) and 11 ITS2 alleles (nine previously defined and two new) were observed. A total of 19 ITS haplotypes, including five novel haplotypes (DEL1r, Edel2, Hr, Adel3 and SYD1a), were observed. The most prevalent type was SYD1g (16.3%), followed by types Ea (11.6%), Ec (9.3%), Eg (6.9%), DEL1r (6.9%), Ne (6.9%) and Ai (6.9%). To detect mixed infection, 30% of the positive isolates were cloned and 4-5 clones were sequenced from each specimen. Cloning and sequencing identified two more haplotypes in addition to the 19 types. Mixed infection was identified in 3 of the 13 cloned samples (23.1%). Upon construction of a haplotype network of 21 haplotypes, type Eg was identified as the most probable ancestral type. The present study is the first study that describes the haplotypes of P. jirovecii based on the ITS gene from India. The study suggests a high diversity of P. jirovecii haplotypes in the population.