Edinburgh Napier University

  The aim of this study was to develop a sensitive and specific method of detecting Toxoplasma gondii in the immunocompromised host which would reduce the need for other tests and would ensure the prompt initiation of the appropriate treatment, the effects of which could be monitored. Such a system would also be of benefit in the
investigation of parasite / host interaction.
Initial work investigated an antigen ELISA and the PCR using two different gene targets CB 1 and P30) to find the most sensitive system. The ELISA was insensitive but both PCR systems were capable of detecting parasite in blood, lymph and tissue samples from experimentally infected sheep. The B 1 PCR detected parasite earlier and over a significantly longer period than the P30 PCR, this greater sensitivity being due to the higher copy number of the B 1 gene.
The PCR was applied to samples from patients with AIDS with the aim of finding an ideal sample for the diagnosis of infection. Parasite was detected in blood up to a month prior to clinical signs of infection, and therefore blood samples are ideal for monitoring
patients at risk of recrudescence of a chronic infection. This result indicates that recrudescence is not due to local reactivation, but is due to a more widespread parasitaemia. However, as parasitaemia was shown to be transient in cases of recrudescence, sampling time may be critical. Parasite was also detected in urine, biopsy
tissue and post mortem material, but was not detected in CSF.
Dexamethasone was used to create a mouse model of recrudescence in the immunocompromised patient to further investigate interaction between the parasite and host. The PCR detected parasite in blood, brain and heart of chronically infected animals, however the detection rate was significantly higher in groups receiveing immunosuppressive therapy. Dexamethasone treatment mimicked the effects seen in the AIDS population where 30-35% of chronically infected individuals showed clinical signs
of toxoplasmosis. However the PCR may also be detecting latent cysts in tissue samples, and blood samples were occasionally positive without clinical evidence of infection. This could be due to small amounts of parasite circulating intermittently, or to breakdown
products from parasite degradation.
There was therefore a need to differentiate between active and chronic infection, and this was carried out by developing a quantitative PCR based on competitive amplification. A novel Sma I restriction site was created within the P30 gene, and known amounts were co-amplified with samples. The amplified products were then digested with Sma I to differentiate between mutated and T. gondii DNA and the point at which product yield was equalled indicated the amount of original DNA present in the sample. The system was shown to work using human PM samples, and could be adapted to indicate a cut-off point where parasite DNA levels reveal active infection.
In conclusion the B 1 PCR is the method of choice in detecting T. gondii in AIDS patients. Any patient in which active parasite is detected should be treated and closely monitored using the qPCR for any evidence of reactivation.