title: Vittaforma corneae keratitis in southern India: role of a novel duplex PCR.
abstract: Microsporidia are obligate intracellular parasites that infect eukaryotic cells and have emerged as major opportunistic human pathogens. Due to the difficulties in definitive laboratory diagnosis and insufficient knowledge, ocular microsporidiosis is infrequently reported in India. To improve diagnostic facilities, we have developed a novel duplex PCR (dPCR) for the simultaneous identification of both genera and species of isolates with microsporidian aetiology that cause keratitis. The material scraped from the corneas of 12 clinically diagnosed microsporidial keratitis patients was subjected to routine microbiological examinations and molecular diagnosis using a novel dPCR that targeted the small-subunit rRNA gene (SSU-rRNA) of microsporidia and Vittaforma corneae using genus- and species-specific primers. Of the 12 corneal scrapes, 6 showed positive results in smears, while dPCR provided positive amplification with both pan-microsporidial and V. corneae species-specific primers for 9 corneal scrapes. The results were validated by sequencing and blast analysis. The sensitivity of this novel dPCR method was higher than that of conventional microscopy in the diagnosis of corneal microsporidial infection. dPCR with specific primers is potentially more sensitive, specific and depends less on more complicated methods for exact identification of the aetiology of microsporidial keratitis.
authors: Department of Microbiology and Molecular Biology, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Tirunelveli, Tamil Nadu 627 001, India.
authors: M
authors: M
authors: Jayahar Bharathi
authors: Department of Microbiology and Molecular Biology, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Tirunelveli, Tamil Nadu 627 001, India.
authors: N
authors: N
authors: Murugan
authors: Department of Microbiology and Molecular Biology, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Tirunelveli, Tamil Nadu 627 001, India.
authors: G
authors: G
authors: Ramesh Kumar
authors: Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Tirunelveli, Tamil Nadu 627 001, India.
authors: R
authors: R
authors: Ramakrishnan
authors: Department of Cornea and External Eye Diseases, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Tirunelveli, Tamil Nadu 627 001, India.
authors: V
authors: V
authors: Anitha
authors: Postgraduate Department of Microbiology, Sri Paramakalyani College, Alwarkuruchi, Tirunelveli, Tamil Nadu 627412, India.
authors: S
authors: S
authors: Ramesh
mesh_terms: D019411:Clinical Laboratory Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D020459:Genes, rRNA; D006801:Humans; D007194:India / Q000453:epidemiology; D007634:Keratitis / Q000175:diagnosis* / Q000382:microbiology; D016881:Microsporidiosis / Q000175:diagnosis* / Q000382:microbiology; D025202:Molecular Diagnostic Techniques / Q000379:methods; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009172:Mycology / Q000379:methods; D012331:RNA, Fungal / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D021863:Vittaforma / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D004275:DNA, Ribosomal; D012331:RNA, Fungal; D012337:RNA, Ribosomal, 18S