Evaluation of Nested PCR Targeting B1 gene With Four nPCRs (Targeting the B1, SAG2 and SAG1 Gene) for Detection of Toxoplasma gondii Genome in Intra Ocular Fluids From Toxoplasma Retinochoroiditis Patients in a Tertiary Eye Hospital, Chennai, India

Abstract

Purpose: : To evaluate nPCR targeting B1 gene (nucleotide 128-707-primer set I) with 4 other nPCRs (one each targeting B1 (nucleotide 694-887-primer set II), SAG1, 3’ end of SAG2 and 5’ end of SAG2 gene), for detection of T. gondii genome on aqueous humor (AH) and vitreous aspirate (VA) from Toxoplasma retinochoroiditis (TRC) patients

Methods: : DNA extracted from 212 intraocular fluids (112 TRC patients and 100 controls) were included in the study. DNA from all the 97 AH and 15 VA from 112 TRC patients were subjected to nPCR targeting B1 gene (primer set I) and among these, DNA from 24 intra ocular fluids from TRC patients were subjected to each of the other 4 other nPCRs -one each targeting B1 gene (primer set II), SAG1 gene, 3’ end and 5’ end of SAG2 gene respectively. DNA extracted from the AH of 61 control patients undergoing un-complicated cataract surgery and 39 control patients with ocular inflammation of non-Toxoplasma origin were subjected to each of the 5 different nPCRs.

Results: : All the 5 nPCRS were specific to detect T. gondii genome and sensitive to detect DNA from one tachyzoite of T. gondii. T. gondii genome was detected by atleast one of the 5 nPCRs in 40 (35.7%) of 112 TRC patients.T. gondii genome was detected in none of the 100 intra ocular fluids from the controls by all the 5 nPCRs. nPCR targeting B1 gene (primer set I)) was positive in 33 AH and 5 VA from 38 (33.9%) of 112 TRC patients, B1 gene (primer set II) in 2 AH (8.3%) of 24 TRC patients tested, both 3’end of SAG2 and 5’ end of SAG2 in 5 AH (20.8%) of 24 TRC patients respectively, and nPCR targeting SAG1 gene in none of the 24 AH from TRC patients. The sensitivity of nPCR targeting B1 gene (primer set I) was statistically significant (Yates correction - Chi square test; p= 0.028), compared to the other 4 nPCRs.

Keywords: toxoplasmosis • retinochoroiditis • anterior chamber