Background Despite the recent advances in diagnosis, Neisseraia gonorrhoeae (NG) and Chlamydia trachomatis (CT) remain leading cause of STDs worldwide and account for STI's morbidity leading to acquisition of HIV and HPV infection. Coinfection of gonococcus and Chlamydia is reported from various countries. In recent study we reported high rate of infection by CT using an in house developed PCR method. A number of studies also suggest increasing rate of antibiotic resistance in NG. Thus we developed a rapid, specific and cost effective diagnostic method to detect prevalence of co infection by NG and CT. Methodology: (1) Unique gene sequence of CT and NG were amplified from gDNA isolated from endocervical swabs. (2) Validation of in house PCR method using Roche AMPLICOR Micro well plate CT/NG kit. (3) Use of molecular beacon to develop easy visual method. (4) Establishment of multiplex PCR (mPCR) for simultaneous detection of CT and NG.
Results 360 clinical samples were used to validate in house PCR assay. Discrepancy of the samples was resolved by amplifying genes encoding for outer membrane proteins (rmp for NG and ompA for CT). The resolved PPV and NPV were found to be 94% and 99% for CT, 92.0% and 96% for NG. Molecular beacon probe was used which helped in visualisation of PCR product directly in tube using dark reader which also improved the sensitivity of assay. The overall infection rate by NG was 26% and 8.6% in symptomatic and asymptomatic patients while that of CT was 26.3% and 21% respectively. To make the method easy to use in remote areas with minimum laboratory infrastructure, the in house PCR assay was standardised for detection using dry swabs, with crude DNA preparation and stabilisation of reagents at 4C (up to 6 months) was achieved for easy transportation and storage. Using single PCRs, coinfection by CT and NG was found to be 18% in symptomatic (74/410) and 5 % in asymptomatic patients (18/360). We further developed mPCR for simultaneous detection two pathogens. Sensitivity of in house mPCR was found to be 95% when evaluated against PCR for individual pathogen.
Conclusion Detection of both the pathogens in single PCR assay makes it economical both in terms of cost and time. The in house assay is highly sensitive, easy to perform and requires minimum infrastructure as well as technical expertise, making it a better option for routine diagnosis of genital infection in developing countries, which would be of great consequences in disease management.