Use of gene amplification (Polymerase Chain Reaction/ PCR) in the investigation and diagnosis of toxoplasmosis

Background

Gene amplification methods (PCR, LCR, NASBA, etc.) are now used widely in the diagnosis of infectious diseases. Key advantages are their relative speed, the potential to detect very low numbers of pathogens (or, more precisely, specific nucleic acid sequences from pathogens) and the ability to discriminate accurately at the species or sub-species level.

In the case of non-persistent pathogens that are cleared from the body, a positive PCR finding is usually significant. The diagnosis of toxoplasmosis by PCR, however, is complicated by the fact that the parasite persists (principally in heart brain and skeletal muscle in the form of quiescent tissue cysts) for many years after active infection has ceased. Thus, the presence of T. gondii in such tissues does not necessarily equate to active toxoplasmosis.

PCR for T. gondii

A number of studies have been published addressing the potential of PCR in the diagnosis of toxoplasmosis in a range of clinical scenarios including immunocompetent adults, pregnancy, organ transplantation, ocular disease and the immunosuppressed/immunodeficient. In general PCR positive findings in fluid specimens (blood, CSF, amniotic fluid, bronchoalveolar lavage, vitreous humour) generally indicate active infection and, hence, are usually clinically significant. However, the detection of T. gondii in solid tissues such as heart, muscle and brain is of very limited use in confirming active toxoplasma infection since PCR cannot discriminate the active (trophozoite) and quiescent (bradyzoite) forms of the parasite. However, exceptions are the presence of T. gondii in any foetal specimen (which would confirm congenital infection).

Collection and transport of specimens for PCR

Specimens submitted for testing by PCR should be collected exclusively for this purpose and not shared with other tests (e.g., CSF submitted for PCR should not have been handled previously for microscopy, etc.). This is in order to reduce the risk of contamination of specimens with T. gondii-infected material. Further, it should be ensured that equipment used for the collection and handling of specimens is similarly free of extraneous materials that might contain T. gondii.

T. gondii DNA appears to be relatively stable over a period of several days in specimens free from other microbial contamination. Thus, these can usually be transported in the same way as for specimens submitted for serology. However, whole blood specimens may suffer from erythrocyte lysis during prolonged transit or may require fractionation before PCR. For these reasons, this type of specimen should be transported as rapidly as possible, preferably within 24 hours, to the PCR Laboratory .