As in many other fields of laboratory diagnosis of microbiological agents, the introduction of PCR technology has provided the clinician with a new tool, enabling rapid and improved, early and adequate diagnosis of T. gondii infections. Despite the comparatively low prevalence of this parasite in the Nordic countries, the increased number of immunocompromised patients in medical care has extended the demand for rapid and reliable diagnosis of reactivated infection. As an example, the PCR-based diagnosis of T. gondii encephalitis on samples from cerebrospinal fluid (Ostergaard et al. 1993) was found to be the most useful test in comparison to conventional serology and antigen detection (van de Ven E et al. 1991). Furthermore, retinitis caused by T. gondii may be diagnosed by PCR assay of aqueous humour in HIV-infected patients (Danise et al. 1997). One caution that should be raised concerns the difficulties of interpretation of negative PCR results obtained after initiation of treatment of the infection, as was shown in a study of generalized toxoplasma infections in HIV patients (Foudrinier et al. 1996).
Diagnosis of congenital infections caused by T. gondii poses special problems, especially where transmission from the mother is the result of subclinical infection (Thulliez et al. 1992). Improved diagnosis is of particular importance in the newborn child, where early and safe treatment may reduce the sequelae of congenital toxoplasmosis (Thulliez et al. 1992). In this field, PCR has proven superior to other diagnostic methods in a large study of prenatal infections (Hohlfeld et al. 1994). The burden for the clinician of difficult decisions
Table 29.1 Examples of sample materials (X) selected by clinicians for PCR-based diagnosis of T. gondii infection in different clinical conditions
Sample material PBMCCSF Amniotic fluid
Prenatal infection Congenital infection PCR positive/number testeda
Note a Samples were referred to the laboratory for investigation of patients with clinical signs and symptoms of T. gondii infection.
concerning termination of pregnancy or prenatal treatment is somewhat relieved by the increased possibility of reliable diagnosis in these cases (Hohlfeld et al. 1994).
PCR methodology has recently been further improved by the introduction of conventional or real time quantitative PCR analysis of T. gondii DNA (Luo et al. 1997; Lin et al. 2000; Costa et al. 2001). Consistently negative findings in large sample materials from control groups suggest that positive PCR results are of clinical relevance. However, it must be borne in mind that the presence of tissue cysts in very small numbers may be due to latent rather than active infection (Ruskin and Remington 1976). Of AIDS patients who are toxoplasma-seropositive only around 30% will experience reactivation leading to severe disease (Luft and Remington 1992). It is conceivable that the passenger nature of the organism could mislead, and careful interpretation of laboratory results is required. Further diagnostic development will depend on a continuing communication between the clinician and the microbiologist concerning the relevance of PCR findings from different sample material. Examples of body fluids and specimen materials currently being used for PCR-based T. gondii diagnosis are given in Table 29.1. Clinical presentation and severity of disease in the individual may vary due to factors associated with the host, and with the genotype of the infecting parasite. Despite the existence of a well-described sexual cycle in cats, T. gondii appears to reproduce largely clonally in nature, recombination being a rarer event. PCR-restriction polymorphism (PCR-RFLP) analyses at multiple loci indicate that three distinct lineages dominate (Howe and Sibley 1995). Although only limited data are available on strain type and infection in humans (Honore et al. 2000; Grigg et al. 2001b), recent reports based on direct PCR-RFLP of patient samples found no clear correlation between genotype, symptoms, and severity of disease (Fuentes et al. 2001). Direct PCR-RFLP analysis may provide new perspectives for investigation of T. gondii infections, and eventual refinement of diagnostic methods. However, the immediate concern remains provision of a rapid and reliable detection, and identification, of the causative organism, T. gondii. In what is often a potentially blinding, or fatal infection that is amenable to therapy, rapid identification of T. gondii can be crucial.
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