Use of Multicopy and Single Copy Genes for Detection of Pneumocystis jirovecii and Subsequent Analysis of Genotypes of Indian Isolates

Background: Pneumocystis carinii Pneumonia (PCP) caused by an opportunistic agent Pneumocystis jirovecii is one of the most serious respiratory infections in immunocompromised patients including HIV infected individuals. Enormous gaps still exist in understanding the basic biology of the organism and epidemiology of PCP due to nonavailability of an appropriate propagation system. In the present study, we carried out molecular detection of the organism using two multicopy and one single copy gene. Further genotypic study was performed on some of the isolates to assess the genotypes. Methods: From November 2005 to October 2006, 114 different respiratory specimens were collected from 90 patients with various underlying conditions and a clinical suspicion of PCP. PCR was done for three independent gene loci including two multicopy i.e. Major Surface Glycoprotein (MSG) and mitochondrial Large subunit rRNA (mtLSUrRNA) and one single copy, internal transcribed spacer (ITS) region Results: Single round MSG and mtLSUrRNA PCR showed 9 positive isolates. External round of ITS PCR detected 6 cases while only nested round could detect all 9 cases. On applying nested PCR for mtLSUrRNA and ITS PCR we detected 5 additional cases. We further sequenced six isolates for mtLSUrRNA gene. Different genotypes of Pneumocystis were distinguished by identifying polymorphisms at position 85 and 248 of mtLSUrRNA. Two genotypes i.e. type 2 (5/6) and 3, (1/6) were observed among the six isolates. Also, one of our previous isolate showed genotype 1. Conclusion: Thus, among multicopy genes, MSG and mtLSUrRNA appears to be equally sensitive while in case of single copy ITS a nested PCR assay is required. In our present study, genotype 2 was more frequently detected (5/7), followed by type 3 and 1(1/7) and a prospective study with large number of patients is being carried out in our centre to address this issue. To our knowledge this is the first study from South Asia describing genotypes of isolates of P. jirovecii .