Microsporidial keratitis in India: 16S rRNA gene-based PCR assay for diagnosis and species identification of microsporidia in clinical samples

Abstract

Purpose: To evaluate 16S rRNA-based polymerase chain reactions for the detection and species identification of the microsporidia that cause keratitis. Methods: Of the 5892 cases of microbial keratitis seen between September 2002 and December 2005, 31 (0.5%) microscopically diagnosed cases of microsporidial keratitis were included in the test group; 103 patients with nonmicrosporidial keratitis constituted the control group. A 16S rRNA-based pan-microsporidian PCR was chosen for the detection of microsporidian DNA. Species level identification was made using species-specific primer sets of Encephalitozoon spp (E. cuniculi, E. hellem, and E. intestinalis). Sequencing and BLAST analysis of amplicons obtained with pan-microsporidian primers were performed for validation. Results: The corneal scrapings from 26 of 31 cases in the test group and 2 of 103 cases in the control group showed a 250- to 280-bp amplicon in PCR by pan-microsporidian primers (sensitivity of 83% and specificity of 98%). The amplicons of 13 of 26 test group samples were identified by species-specific PCR: E. cuniculi, n = 7 (549 bp); E. hellem; n = 3 (549 bp); E. intestinalis; n = 1 (520 bp). The two cases in the control group were identified to be E. cuniculi. The remaining 15 cases (test group) were confirmed to be Vittaforma corneae by sequencing and BLAST analysis. All species were confirmed by sequencing and database homology comparison. Conclusions: This study is the first to validate PCR-based assays for detection of microsporidial DNA in corneal scrapings. Pan microsporidian PCR can be a useful adjunct with smear examination in the diagnosis of microsporidial keratitis.