PCRbased diagnosis of Toxoplasma gondii strategies and tactics

A general problem with T. gondii diagnosis is that active disease may be reflected by the presence of only a few tachyzoites in some types of specimens. Although PCR methods facilitate the detection of as little as one or a few parasites present in such specimens, it could be erroneous to rely only on the high sensitivity of the test. First, the Poisson distribution of tachyzoites in a fluid specimen implies that at low tachyzoite densities there is a significant probability that negative results are obtained due to an uneven distribution. Moreover, special care is needed to ensure that the few tachyzoites contained in such specimens are not non-specifically adsorbed or degraded during transport to the laboratory. With this background, a safer strategy would be to include examination of specimens from locations expected to contain higher concentrations of T. gondii, such as lymph nodes, or to analyse several portions, preferably over a time interval, from easily obtained body fluids such as PBMC, before ruling out the diagnosis. As these infections may persist and cause symptoms over many weeks and months, repeated diagnostic efforts are recommended in analogy with the search for bacterial pathogens by repeated blood culture sampling from patients with prolonged fever of unknown origin. The introduction of several PCR methods in different laboratories for T. gondii diagnosis raises the important question of which method represents the optimal technology for the Nordic countries. However, with the documented high sensitivity and specificity of different methods, we see several advantages in diversity within this diagnostic field (Lebech and Petersen, 1992; Stray-Pedersen and Jenum, 1992; Ostergaard et al. 1993; Lappalainen et al. 1995; Guy et al. 1996). Just as for PCR-based detection of other microbial agents, where we have some experience from the field of herpesvirus infections, reviewed in (Bergstrom et al. 1995), there is probably no such thing as a 'gold standard' setup. Thus, methods appearing as excellent in one laboratory can perform poorly in another, and vice versa. In addition, protozoa, as other microbial agents, are under constant evolution, and therefore the use of different independent PCR primer systems could be an advantage and help in discovering emerging strains. Although recent population genetic studies identified a very limited number of T. gondii genotypes in nature, recombination may occur even though a rare event (Grigg et al. 2001a,b). Studies of genotype, associated human clinical disease, and immunocompetence in different patient groups, may provide new perspectives for investigation of toxoplasma disease, and eventual refinement of diagnostic methods. But the essential requirement of a reliable, well-proven detection system, such as the P30 or B1 PCR, for rapid laboratory diagnosis of T. gondii infections, and an awareness of the importance of maintenance of good liaison between clinician and laboratory, cannot be over emphasized.

Standardization programmes of Nordic PCR-based diagnosis of T. gondii infections should therefore focus on the exchange of specimens and standards rather than striving towards a consensus PCR protocol. Furthermore, this information exchange should include sharing of experiences concerning suitable sample materials as well as optimal time points and conditions for sampling. To conclude, a strategy combining the advantages of PCR methods with a sharing of experience between different laboratories and the clinicians seems especially suitable for the improvement of T. gondii diagnosis in a low-prevalence area with a well-organized health care system.

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