Nucleic acid amplification Chlamydial infections are frequently asymptomatic. - Confirmation of chlamydial infection usually depends on taking an appropriate clinical sample .from the patient ,followed by the direct detection of the organism using a suitable laboratory-based diagnostic test. The demonstration of chlamydial antibodies (serology) in an individual is rarely diagnostic. For many years, the optimum method of confirming the presence of chlamydial infection was the growth of the infecting organism in cell culture and the demonstration of characteristic chlamydial inclusions. - However, this method necessitated the availability of good transpo and cold-storage facilities in order to maintain the bility of the organism prior to inoculation. - Moreover growth and isolation of the organisms in cell culture was relatively tedious and difficult to quality control. - Chlamydial cell culture ,facilities were available to relatively few clinical centres. - It is now widely recognized that cell culture techniques were, at best, only 60 to 80% sensitive. A key advance in the laboratory diagnosis of chlamydial infections has been the development of non-bility dependent tests which place less demand on specimen transpo. The first of these tests were the chlamydial antigen detection tests, which relied, either on The direct detection of chlamydial elementary bodies in clinical material using fluorochrome-labelled, chlamydial specific, monoclonal antibodies. Direct immunofluorescence - Capture and detection of chlamydial antigen in an extract of clinical material using enzyme immunoassay based procedures. These methods are still appropriate in some settings and they remain in widespread use. However, they are gradually being superseded by newer, methods based on the detection of chlamydial nucleic acid, either by -- Direct hybridization or by - Nucleic acid amplification The latter use a variety of amplification reactions including the: ? - Polymerase chain reaction - Ligase chain reaction - Strand displacement amplification or transcription mediated amplification. Nucleic acid-based methods generally offer superior sensitivity and specificity to the antigen detection tests, but at greater cost and a greater requirement for trained staff. A second major advance has been the recognition that cervical or urethral specimens requiring invasive genital sampling are by no means essential for the laboratory diagnosis of chlamydial infection. Non invasive and or self collected samples which may be adequate include male and ,female urine, various vulval-introital samples or vaginal fluid expressed from a tampon. More on chlamydial diagnosis Chlamydia is an obligate intracellular parasite. It cannot survive outside its host i.e. it cannot grow on cell free media. - Up until the early 1980s, the main method of confirming a diagnosis of chlamydial infection, was the inoculation of clinical material into animals, embryonated hens eggs or (usually) tissue culture cells and the demonstration of characteristic chlamydial inclusions. Generally speaking inclusions were demonstrated in cell culture ? - Either by staining with iodine - By staining with Giemsa stain - Fluorochrome-labelled poly - or mono-clonal antibody - Enzyme immunohistochemistry The usual tissue culture cells were - - HeLa 229, - L434 mouse fibroblasts or C'. trachomatis and C. psittaci; - McCoy cells - Buffalo Green Monkey kidney cells (C. psittaci and - HL or Hep2 cells C. pneumoniae) Growth of the organisms was facilitated by the use of ? - Antimetabolites directed against the host cell (cyclohexitnide; emetine or ntitomycin C) or, - By the use of charged anionic polymers such as Poly - L - lysine or DEAR dextran for C trachomatis TRIO biovar. Compounds like polyethylene glycol or high energy glucose 6 phosphate also aided the growth of some chlamydiae.