Evaluation of Nested PCR Against Witmer Desmonts Coefficient on Aqueous Humor for Diagnosis of Ocular Toxoplasmosis in HIV Positive Patients and Genotyping of Toxoplasma Gondii

Abstract

Purpose: : The present study was undertaken to evaluate nested polymerase chain reaction (nPCR) targeting B1 gene of Toxoplasma gondii (T. gondii) with Witmer Desmonts Coefficient (WDC) technique on aqueous humor (AH) of HIV positive, clinically suspected Toxoplasma retinochoroiditis (TRC) patients and to genotype T. gondii by nPCR- RFLP of SAG2 gene and DNA sequence of B1 gene.

Methods: : Serum and AH were collected from 34 HIV positive patients including 23 TRC patients and 11 disease controls. Standard method of WDC and standardized nPCR targeting B1 gene (primer set 1) was applied on the 34 AH. AH samples positive by nPCR of B1 gene (primer set 1) were further analysed for genotyping of T. gondii by nPCR-RFLP targeting SAG2 gene and DNA sequencing of amplified products of nPCR targeting B1 gene (primer set 2).

Results: : Among the 23 AH of TRC patients, nPCR (primer set 1) was positive in 52.1%, and WDC in 43.5% (Statistically not significant; p= 0.1769). nPCR and WDC were negative in 11 AH from control patients. Among the 12 nPCR positive AH, Genotype I of T. gondii was identified in 3 (25%) by nPCR-RFLP targeting SAG2 gene and only two were confirmed to be of genotype I with unique sequence (Accession no: EU334492; EU183361) by DNA sequence of amplified products of nPCR targeting B1 gene (primer set 2) .

Conclusions: : nPCR and WDC are reliable techniques for laboratory diagnosis of TRC in HIV positive patients. This is the first study in India to demonstrate that genotyping of T. gondii in ocular toxoplasmosis patients is possible by nPCR based RFLP/ DNA sequencing techniques targeting SAG2 gene and B1 gene.

Keywords: toxoplasmosis • retinochoroiditis • AIDS/HIV