TITLE: Standardization of nucleic acid based amplification techniques in detection and identification of fungi in clinical specimens from clinically suspected cases of infectious keratitis and endophthalmitis ( SR/SO/HS-63/2003)

Principle Investigator : Dr. K. Lily Therese

OBJECTIVES:

•  To optimize and apply a multiplex Polymerase Chain reaction (mPCR) targeting 18SrRNA, ITS and 28SrRNA for detection of panfungal genome from clinically suspected cases of infectious keratitis and endophthalmitis.

•  To identify the non-sporulating moulds (NSM) by PCR based DNA sequencing and PCR-RFLP.

•  To apply PCR, PCR based Restriction Fragment length Polymorphism (PCR-RFLP) and DNA sequencing to identify Fusarium species.

I. Optimization of mPCR: mPCR was optimized targeting the 18SrRNA, ITS and 28SrRNA region. The sensitivity of mPCR was 1 picogram and it was specific only the for fungal DNA.. mPCR detected the presence of panfungal genome in 10 corneal scrapings and 10 intraocular fluids in 40 ocular specimens processed .

II. PCR based Restriction Fragment length Polymorphism (PCR-RFLP) and DNA sequencing to identify Fusarium species.

Ten isolates of Fusarium species were identified based on colony morphology, microscopic morphology and PCR-RFLP targeting ITS region.

III. PCR based RFLP and DNA sequencing targeting ITS region for the identification of NSM to species level.

A total of 10 NSM isolates were subjected to DNA sequencing. The results of DNA sequencing identified 5 isolates as Botryosphaeria rhodina , 2 as Pythium insidiosum , 1 Macrophomina phaseolina , 1 Rhizoctonia bataticola . The 3 genera consisting of Botryosphaeria, Macrophomina, Rhizoctonia were found to be the potential aetiological agents causing fungal keratitis in Indian population. The 5 isolates of Botryosphaeria rhodina recovered from the same patient exhibited the same type of nucleotide polymorphisms in the ITS region.