Molecular diagnosis of infection with Toxoplasma gondii in immunocompromised patients

The protozoan parasite Toxoplasma gondii infects more than a billion people worldwide. The parasite is generally divided into three clonal lineages, denoted type I, II and III. Primary infections in humans are usually asymptomatic or characterized by non-specific symptoms, and are followed by latent chronic infection, which is normally harmless to the host. However, if the immune system is suppressed, as in bone marrow transplantation (BMT) or human immunodeficiency virus (HIV) infection, reactivated toxoplasmosis or primary infection may cause severe disease. Molecular methods are the primary tools to diagnose toxoplasmosis in immunocompromised patients. The molecular diagnosis would benefit from standardization and a risk profile to identify patients who should be tested for toxoplasmosis by the polymerase chain reaction (PCR). The present thesis shows that real-time PCR preceded by manual or automated DNA extraction of pure parasites could be used to detect T. gondii DNA corresponding to one parasite genome in a reaction volume. Different detection limits were however obtained when whole blood samples were spiked with parasites, and analyzed with conventional PCR, nested PCR oligochromatography, real-time PCR SYBR green, or real-time PCR TaqMan. The results show that DNA extraction methods and PCR assays may perform differently depending on the type of sample. A comparative study of two real-time PCR targets, a 200-300-fold repeated 529 bp element, and the 35-fold repeated B1 gene, showed that T. gondii DNA was detected more sensitively and more accurately when a more repeated PCR target was used. Furthermore, the performance of real-time PCR targeting the 529 bp element was not affected when an internal amplification control was included. A rapid and highly reproducible Pyrosequencing assay was developed, which is a promising method for routine use to discriminate between type I, II and III T. gondii strains in clinical samples. Identification of T. gondii strains may help to increase our knowledge about the significance of the genotypes in human toxoplasmosis. A prospective study of the magnitude of toxoplasmosis in BMT recipients, performed by PCR and real-time PCR analysis of peripheral blood samples, showed that it was not possible to identify a specific risk profile associated with an increased risk for toxoplasmosis. A retrospective study of pulmonary toxoplasmosis in immunocompromised HIV patients, performed by real-time PCR analysis of bronchoalveolar lavage samples, confirmed that a particular risk profile could not be identified. However, it seems important that surveillance by PCR is initiated immediately after allogeneic BMT in seropositive patients, and the conclusion was made that a PCR test for T. gondii should be performed if pulmonary symptoms of unknown aetiology occur in immunocompromised HIV positive patients. After review of medical records we suggest that toxoplasmosis may be suspected if ocular infections of unknown aetiology occur in seropositive allogeneic BMT patients and HIV patients, and that asymptomatic and symptomatic toxoplasmosis may occur despite trimethoprim/sulfamethoxazole prophylaxis. We suggest that monitoring of seropositive immunocompromised patients using molecular methods may serve multiple purposes; i) to diagnose toxoplasmosis at an early stage to facilitate effective treatment, ii) to correlate the parasitic burden to clinical symptoms, iii) to further investigate the significance of the genotype of the parasite, and iv) to evaluate the ability of prophylactic regimens to prevent reactivations and full-blown toxoplasmosis. LIST OF PUBLICATIONS I. Edvinsson B, Jalal S, Nord CE, Pedersen BS and Evengård B, DNA extraction and PCR assays for detection of Toxoplasma gondii. APMIS 2004 Jun;112(6):342-8. II. Edvinsson B, Lappalainen M and Evengård B, Real-time PCR targeting a 529-bp repeat element for diagnosis of toxoplasmosis. Clin Microbiol Infect 2006 Feb;12(2):131-6. III. Edvinsson B, Dardé ML, Pelloux H and Evengård B, Rapid genotyping of Toxoplasma gondii by Pyrosequencing. Submitted to Clin Microbiol Infect IV. Edvinsson B, Lundquist J, Ljungman P, Ringdén O and Evengård B, A prospective study of diagnosis of Toxoplasma gondii infection after bone marrow transplantation. Manuscript. V. Petersen E, Edvinsson B, Lundgren B, Benfield T and Evengård B, Diagnosis of pulmonary infection with Toxoplasma gondii in immunocompromised HIV-positive patients by real-time PCR. Eur J Clin Microbiol Infect D 2006 Jun;25(6):401-4.