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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) References Ahmadian A, Ehn M, Hober S (2006). Pyrosequencing: history, biochemistry and future. Clin Chim Acta. 63:83-94. Beghetto E, Spadoni A, Bruno L, Buffolano W, Gargano N (2006). Chimeric antigens of Toxoplasma gondii: toward standardization of toxoplasmosis serodiagnosis using recombinant products. J Clin Microbiol 44: 2133-40. Bohne W, Parmley SF, Young S, Gross U(1996). Toxoplasma gondii. In: Gross U (ed) Current Topics in Microbiology and Immunology. Berlin, Springer-Verlag; 219: 81–91. Botterel F, Ichai, P, Feray, C Bouree, P, Saliba, F, Tur Raspa R et al (2002). Disseminated Toxoplasmosis, resulting from infection of allograft, after orthotopic liver transplantation: usefulness of quantitative PCR. J Clin Microbiol 5: 1648–1650. Bretagne S, Costa, JM Foulet F, Iabot Lestang L, Baud et al (2000). Prospective study of Toxoplasma reactivation by polymerase chain reaction in allogenic stem-cell transplant recipient. Transpl Infect Dis 2: 127–132. Buchbinder S, Blatz R, Rodloff AC (2003). Comparison of Realtime PCR detection methods for B1 and P30 genes of Toxoplasma gondii. Diagn Microbiol Infect Dis 45: 269–271. Burg JL, Grover CM, Pouletty P, Boothroyd JC (1989). Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. J Clin Microbiol; 27: 1787–1792. Cazenave J, Cheyrou A, Blouin P, Johnson AM, Begueret J (1991).
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Advanced laboratory techniques for diagnosing Toxoplasma gondii encephalitis in AIDS patients: significance of intrathecal production and comparison with PCR and ECLwestern blotting. J Neuroimmunol; 92:29-37. Contini C, Seraceni S, Cultrera R (1999). Different PCR systems to detect Toxoplasma gondii tachyzoites or bradyzoites in clinical specimens from patients with and without overt disease. J Eukaryot Microbiol; 46: 77S–78S. Contini C, Seraceni S, Cultrera R, Incorvaia C, Sebastiani A, Picot S (2005). Evaluation of a Real-time PCR-based assay using the light cycler system for detection of Toxoplasma gondii bradyzoite genes in blood specimens from patients with toxoplasmic retinochoroiditis. Int J Parasitol; 35:275-83. Costa JM, Pautas C, Ernault P, Foulet F, Cordonnier C, Bretagne S (2000). Real-time PCR for diagnosis and follow-up of Toxoplasma reactivation after allogeneneic stem cell transplantation using fluorescence resonance energy transfer hybridization probes. 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Toxoplasma myocarditis presenting as myocardial infarction. Indian J Med Sci; 61:218- 20. Edvinsson B, Lappalainen M, Evengård B; ESCMID Study Group for Toxoplasmosis (2006). Real-time PCR targeting a 529-bp repeat element for diagnosis of toxoplasmosis. Clin Microbiol Infect;12:131-6. Eggers C, Gross U, Klinker H, Schalke B, Stellbrink HJ, Kunze K (1995). Limited value of cerebrospinal fluid for direct detection of Toxoplasma gondii in toxoplasmic encephalitis associated with AIDS. J Neurol; 242: 644–649. 49 Fauci AS (1984). Immunologic abnormalities in the acquired immunodeficiency syndrome (AIDS). Clin Res; 32:491-9. Garly M, Eskild P, Petersen C, Lundgren JD, Gerstoft J (1997). Toxoplasmosis in Danish AIDS patients. Scand J Infect Dis; 29: 597- 600 Genot S, Franck J, Forel JM, Rebaudet S, Ajzenberg D, de Paula AM, et al (2007). Severe Toxoplasma gondii I/III recombinantgenotype encephalitis in a human immunodeficiency virus patient. J Clin Microbiol; 45:3138-40. 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Page 31 and 32: Ferry along the Potomac River. The
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Page 46 and 47: 42 References Ades AE, Gilbert R, T
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Page 64 and 65: 60 (Kijlstra et al., 2006). Regardi
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98 test, whereas for the remaining
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100 pyriproxyfen is twice as biolog
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for what concern the time of biting
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fall recorded in the considered per
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SIMPOSIO 4 IL RUOLO DELLA RICERCA N
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136 Program financed by ANTIMAL. S.
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138 A. della Torre et al. - Researc
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E. Schwarzer et al. - Hemozoin and
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148 geographic distribution in sub-
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150 K.E., Beldjord, C., Nagel, R.L.
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Parassitologia 50, 2008 Angelini P.
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