Laboratory Diagnosis of Mycoplasma Infections
1. Culture ;- M pneumoniae isolation is performed routinely in only a few laboratories. The most common method is to inoculate throat swabs (in virus transport medium) or sputum (mixed with an equal volume of N-acetyl cysteine to promote mucolysis) on to solid and liquid media. Plates are incubated at 37oC in 5% CO2 in nitrogen and examined at 3-4 day intervals for 3 weeks. Colonies are subcultured and confirmed as M pneumoniae by means by growth inhibition tests employing filter paper discs impregnated with M pneumoniae antiserum. Broth cultures are incubated at 37oC for 3 weeks. The disadvantage of isolation is that culture and identification may take 10-14 days. Isolation on its own is not a wholly reliable diagnostic procedure since M pneumoniae can be isolated from symptom-free individuals and is known sometimes to exist as an commensal organism in man.
2. Antigen Detection ;- Several techniques have been used to demonstrate M pneumoniae antigens in tissue or body fluids such as immunofluorescence and ELISAs. These ELISAs detected M pneumoniae in 90% of the samples from which the organism was isolated and M pneumoniae antigen was found in 43% of samples from patients with serological evidence of recent infection but from which M pneumoniae was not cultured. DNA probes have been developed for the detection of M pneumoniae DNA in clinical specimens. Although the probe method is sensitive, specific and rapid, it is labour-intensive, expensive.
3. Antibody Detection ;- Many methods have been employed for the detecting M pneumoniae antibodies in human serum. Methods used for the detection of rising titres of IgG are suitable for patients of all ages. However, methods used for detecting IgM are more suitable for use in younger patients, since IgM is found less frequently in patients experiencing re-infection (hence older patients).
CFT is the mainstay of routine laboratory diagnosis of M pneumoniae infections. However, antibodies may not be detected by this method 7-10 days after the onset of symptoms and not all culture-positive patients develop CF antibody or a significant rise in titre. It is also very difficult to determine the significance of CFT titres obtained with single samples of serum, unless they are very high. Such high titres can be found many months after infection and so particularly in the months following periods of high incidence of M pneumoniae infection. Demonstration of a fourfold or greater rise in antibody titre is required to be reasonably sure of the diagnosis.