The application of molecular technology in the study of human Chlamydia trachomatis infections

Chlamydia trachomatis infects the epithelial surfaces of the conjunctivae and genital tract, causing trachoma and a variety of sexually transmitted diseases. Failure of diagnosis and treatment results in persistent infection and severe complication such as blindness, tubal factor infertility and ectopic pregnancy. The objective of the work described in this Thesis was to develop improved PCR tests for chlamydia for use in studies on the immunobiology of human infections. A PCR test based on the plasmid gene was developed and tested against 1,364 conjunctival swabs from the villagers of trachoma endemic area in the West Africa. Genotyping PCR for ocular strains of C.trachomatis was developed using the drop-in, drop-out PCR and fluorescently-labelled primers. Ocular strain variants were identified by direct sequencing of the VS-I and VS-II of MOMP gene. An important aspect of MOMP sequencing was to establish if there was significant antigenic variation in the surface exposed loops on MOMP which react with protective monoclonal antibodies. This information is essential for the design of MOMP based vaccines A PCR test based on the 60 kDa cysteine rich protein of chlamydia used the advance technique of drop-in, drop-out PCR. The system was devised to detect and identify infections caused by C.trachomatis, C.psittaci and C.pneumoniae in a single tube PCR reaction. The test was successful in the diagnosing of chlamydial pneumonia in bronchoalveolar lavage fluid from AIDS patients and detected C.trachomatis in women with laparoscopically proven salpingitis. A novel quantitative PCR was developed. This single tube quantitative PCR comprised 5 standards differing in size and concentration plus the unknown clinical sample DNA; these were co-amplified using a single set of primers. The PCR products generated from the fluorescently-labelled primer were analysed using Automated DNA Sequencer and GENESCAN. The number of chlamydial plasmid in the patient sample was calculated from the standard curve plotted between the number of plasmid copies and the peak area of the different standards.