impaginato piccolo - Società Italiana di Parassitologia (SoIPa)

48
C. Contini - management of toxoplasmosis in immunocompromised patients
depend on several factors including i) the lack of standardized
reagents and protocols for DNA extraction, ii)
the amplification of DNA fragments of different size,
iii) the inadequate storage of the clinical sample (i.e
CSF), and iv) the time elapsing between the start of
specific therapy and CSF collection which often affects
PCR reproducibility and makes comparison of results
difficult. External and internal amplification quality
controls have shown to be helpful tools during development
and validation of PCR assays in diagnosis of toxoplasmosis.
An interesting fragment, the 529-bp sequence, which
has over 300 copies in the genome, has demonstrated
to be specific for T. gondii (Homan WL et al., 2000)
and to improve also sensitivity and accuracy of realtime
PCR (Edvinsson B et al., 2006). A comparison of
methods using the B1 and 529-bp sequences with realtime
PCR revealed a ten-fold improvement in sensitivity
when the 529-bp sequence was used (Reischl U et
al., 2003).
Repetitive sequences including mobile genetic elements
(MGEs) and other single-copy sequences, including the
SAG1, SAG2, SAG3, SAG4 and GRA4 genes (Terry
RS et al., 2001, Rinder H et al., 1995, Meisel R et al.,
1996), have been used as PCR targets in research laboratories.
Their efficacy in the clinical setting needs
however to be further verified in large size studies.
In more recent years, Real-time PCR-based techniques
have been employed for quantitative T. gondii DNA
detection in clinical specimens. Several variations of
the assay have improved sensitivity or specificity and
seem to account for the usefulness of this technique for
laboratory diagnosis. Targets such as the B1 and P-30
(SAG-1) and ribosomal DNA, have shown to be potential
candidates to assure quality for clinical diagnosis of
toxoplasmosis (Bretagne S et al., 2000; Kupferschmidt
O et al., 2001, Hierl H et al., 2004). T. gondii has been
detected by various sequence detection systems including
Light-Cycler (LC) in different human body fluids or
tissue samples from transplant patients, with probes
targeting different genomic fragments. The results varied
in sensitivity (detection limit from 100 to 10 parasites/ml)
and reproducibility (Costa JM et al., 2000;
Lin MH et al., 2000; Kupferschmidt O et al., 2001;
Jones CD et al., 2000; Botterel F et al., 2002;
Buchbinder S et al., 2003).
Although real-Time PCR has been developed since
2000, like many molecular “in-house” assays, this technique
suffers from the absence of an accepted standard
method that allows an optimal comparison of sensitivity
and specificity among the various laboratories.
Recently, we developed a highly sensitive Real-time
PCR which employs LC to detect and quantify T. gondii
B1 and SAG-4, MAG-1 genes in PBMC specimens from
subjects with clinically suspected toxoplasmic
retinochoroiditis, demonstrating that the combination
of blood-real-time PCR with specific genes could
assume an important value to accurately measure the
parasite DNA load and its levels in different moments
of infection, particularly during the course of therapy
(Contini C et al., 2005). The combination of blood-LC-
PCR with these specific genes has demonstrated to be
also useful in transplant patients (data not published).
An important step in the diagnosis of toxoplasmosis
also involves the identification of the genetic group of
T. gondii. In this setting, pyrosequencing has emerged a
suitable technique to discriminate between T. gondii
genotypes in clinical samples (Ahamadian A et al,
2006). Three main clonal lineages known as types I, II
and III, which differ in virulence and epidemiological
pattern of occurrence T. gondii have been genetically
typed (Sibley LD et al., 1992; Howe DK et al., 1995,
Genot JCM et al., 2007). This suggests an influence of
the parasite genotype on disease expression in immunocompromised
patients. The potential correlation
between genotype and disease pattern may thus have
an important impact for the clinicians.
Molecular techniques have undoubtedly become a
major tool in toxoplasmosis diagnosis. Further studies
with large numbers of patients, based on direct analysis
of genetic material by precise methods such as realtime
PCR and more systematic external quality control
and DNA sequencing, are required for a greater understanding
of this important pathogen and also to optimize
the diagnostic approach before they are implemented
as routine methods.
Acknowledgements
This work was in part supported by funds of MIUR (2005-2007)
and Fondazione Cassa di Risparmio di Cento (CARICE) e Ferrara
(CARIFE) (2007-2008)
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