Diagnostic tool development using advanced techniques in biotechnology for microbial keratitis

Abstract

Purpose: To study the demographic characteristics, associated factors, causative agents, of infectious keratitis and develop a diagnostic tool to aid easy diagnosis of keratitis.Methods: Corneal scrapes were collected and subjected to culture, microscopy, considering age, occupation, geographical, Frequency of predisposing ocular conditions, antibiotic susceptibility test, resistance patterns test, drug sensitivity and 16s r-DNAas well as 18s r-DNA based identification was performed and submitted to data bank with accession number. The 16S rDNA sequences of the individual bacteria and fungi were used for a universal primer design and thereby multiplex PCR can be performed.Results: A total of 250 consecutive patients with infective keratitis were evaluated, of which 77 (30.8%) were found to be of bacterial, 67 (26.8%) were fungal, 16 (6.4%) were both fungal and bacterial, and the remaining 90 (36%) were found to be culture negative. Contact lens wear was the main risk factor (80.8%). Ocular surface disease (23.6%), ocular trauma (14.8%), corneal surgery (4.4%) and corneal suture (6.4%) of cases were found in corneal ulcers. Most community acquired bacterial and fungal ulcers resolve with appropriate treatment. 64% of the infections involved positive cultures and 36% involved negative cultures, were found in polymicrobial mode of infection. Fusarium spp. (32.75%) was the most predominant species followed by Aspergillus sp. (20.68%) was found in fungal corneal ulcers and also Staphylococcus sp. was the most common bacteria found in bacterial cultures. The 16S rDNA sequence of the bacteria and fungi cultured from the isolates of the corneal scrapping were performed and the genes were submitted in Genbank.The primers designed for bacteria and fungi gave good results in the multiplex PCR carried out and we suggest this can be used as a diagnostic tool for keratitis.