| Abstract|| |
Background: Toxoplasma gondii infection, common parasitic zoonoses, is an important cause of spontaneous abortion, mental retardation, encephalitis, ocular disease and death worldwide. Today the major diagnostic techniques for the toxoplasmosis are serological assays, but its have many limitations. Aim : The goal in this study is to improve the diagnostic accuracy of T. gondii infection, using direct (Real Time PCR) and indirect (IgM, IgA, IgG and IgG avidity) diagnostic techniques. Materials and Methods: In the period between 2007 and 2008, 96 non consecutive different clinical samples (38 blood, 40 amniotic fluids, 8 cerebrospinal fluids, 10 vitreous humors) and 96 sera have been studied simultaneously through molecular biology and serological techniques. Results: Direct and indirect diagnostic techniques used in this study for laboratory diagnosis of T. gondii infection were always concordant. Conclusions : The high correlation between direct and indirect diagnostic techniques exhibit that serologic techniques are accurate diagnostic assays as screening test in laboratory diagnosis of toxoplasmosis.
Keywords: T. gondii, Toxoplasmosis diagnosis, Antibodies, Serological techniques, Real Time PCR
|How to cite this article:|
Pignanelli S. Laboratory diagnosis of Toxoplasma gondii infection with direct and indirect diagnostic techniques. Indian J Pathol Microbiol 2011;54:786-9
|How to cite this URL:|
Pignanelli S. Laboratory diagnosis of Toxoplasma gondii infection with direct and indirect diagnostic techniques. Indian J Pathol Microbiol [serial online] 2011 [cited 2020 Sep 18];54:786-9. Available from: http://www.ijpmonline.org/text.asp?2011/54/4/786/91503
| Introduction|| |
Toxoplasma gondii is an obligate intracellular apicomplexan parasite with a high distribution in the world. The prevalence of this parasite, in humans, is between 15-85% in the world, depending on age and geographical location.  Often the infection is characterized by non-specific symptoms with the presence of cysts that may remain in latent form in many organs (e.g. muscle, brain etc.). The infection, in immunocompetent individuals, is usually asymptomatic or revealed in 10-20% of cases lymphadenopathy.  However, much less frequently, in immunocompetent individuals severe acquired forms, such as pulmonary forms or chorioretinitis have been described in other study. , When acquired congenitally or as a consequence of reactivation in immunocompromised patients, toxoplasmosis can be life-threatening. In immunocompromised patients it represents the commonest opportunistic infection with clinic neurological complications between 39-70%.  Toxoplasmic infection during pregnancy, instead, may lead to severe, if not fatal, infection of the fetus. The clinical severity of this parasitic infection is gestational time-dependent. A high emphasis is placed on preventive measures, on early etiological diagnosis and appropriate therapy of this infection in order to prevent these severe clinical complications of this zoonosis. Still today the major diagnostic techniques for the toxoplasmosis are serological techniques, despite many limitations.  They fail to detect specific antibodies anti-T. gondii during the active phase of the parasitic infection, because these antibodies are not produced until before several weeks of parasitemia. Therefore, for e.g. the high risk of congenital toxoplasmosis of a fetus may be undetected, because the pregnant mother might to be test negative during the active phase of T. gondii infection. Furthermore, the test may fail to detect T. gondii infection in immunocompromised patients, because the titer of specific antibodies anti-T. gondii may fail to rise in this patient. Furthermore, even if IgM antibodies anti-T. gondii are usually considered a specific marker of recent infection, unfortunately, during T. gondii infection the IgM can persist for long periods, even for months or, in cases more rare, for years. , The antibody avidity has been the subject of several studies in symptomatic subjects and in seroconversion. , The time taken to highlight the antibody avidity is different depending on the method used, although generally it is accepted that a value of high avidity excludes acquisition of infection with T. gondii 3-5 months earlier. Yet values of low or intermediate avidity can persist for more than a year and therefore this fact alone can not suffice for lay diagnosis of recent infection. , Even antibiotic treatments may alter the aging IgG antibody response; in literature are present data on the effect of discordant therapy compared to antibody avidity. , At the same time IgM anti-T. gondii, the IgA can persist for more than a year after the acute phase. For these reasons, a more accurate diagnostic approach is needed to provide a more rapid and a more reliable detection of this pathogen. Several PCR techniques have been developed for the diagnosis of toxoplasmosis, using various clinical specimens (e.g. amniotic fluid, blood, cerebrospinal fluid, etc.) and were tested in various scientific studies. ,, Undoubtedly the serological screening in pregnant women seems to be characterized by data comforting of sensitivity and specificity , (according to the kit used), also it is a simple and low cost test. The objective of this study is to apply at the diagnosis of toxoplasmic infection of direct and indirect diagnostic techniques to evaluate a more accurate and rapid results.
| Materials and Methods|| |
Between 2007-2008, 96 non consecutive different clinical samples (38 blood, 40 amniotic fluids, cerebro-spinal fluids 8, 10 vitreous humors) and 96 sera have been studied. The clinical specimens were sent in our laboratory from different departments: 38.4% from surgery, 24.5% from obstetrics and gynaecology, 13.2% from hemato-oncology, 8.2% from infectious disease, 6.3% from ophthalmology, 6.3% from resuscitation, 3.1% from dialysis. The blood samples came from immunocompromised patients waiting for surgery, the amniotic fluid samples from pregnant women with borderline or positive serological screening for T. gondii, while the vitreous humor and cerebrospinal fluid samples from patients in clinical suspicion of meningitis or retino-choroiditis. Moreover in suspicion of ocular disease, the patient was negative for antibody detection of HSV-1, VZV, CMV, Rubella and Syphilis.
Detection of IgG and IgM antibodies for T. gondii was carried out with the kit Enzygnost Toxoplasmosis IgG and IgM (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). Specific toxoplasmic IgA was obtained by Enzywell Toxoplasma IgA (Diesse, Monteriggioni, Siena, Italy). All serological assays were performed on automated system Genesis RSP 200/BEP III (Siemens Healthcare Diagnostics). Through the methods described have been studied a total of 96 sera (one for each patient). Border-Line or positive IgM results were confirmed by Vidas Toxo IgM (bioMérieux, Marcy l'Etoile, France). Finally, IgG avidity was performed by Vidas Toxo IgG Avidity (bioMérieux, Marcy l'Etoile, France). All assays were performed according to the manufacturer's instructions. ,,,, Performance characteristics and diagnostic value of these assays versus other have been described in literature. ,,, Sensitivity and specificity of the kit used to detect IgA antibodies were respectively 93% and 98%, as described in manufacturer's package insert. Serological diagnosis of acute T. gondii infection was based on the following criteria: detection of specific IgM antibodies (seroconversion) or demonstration of a significant increase in specific IgG antibodies (four fold or greater rise in the titre), or both.  The IgG avidity techniques were utilized only in pregnant women with following serological criteria: simultaneous positivity IgM and IgG.
Extraction of Genetic Material
The DNA extraction was performed using the automated system NucliSens easyMag (bioMérieux, Marcy l'Etoile, France), according to the manufacturer's instructions.  The quantity of clinical specimens used for the extraction was as follows: 1000 μl of amniotic fluid, 500 μl of blood, 500 μl of cerebrospinal fluid, and 500 μl of vitreus humor.
The LightCycler T. gondii LCSet (Roche Molecular Diagnostics, Mannheim, Germany) is a test specifically adapted for Real Time PCR (RT PCR) in glass capillaries, using the LightCycler Instrument and Hybridization Probes for identification of T. gondii in research samples. The Hybridization Probes consist of two different oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the PCR cycle. One probe is labeled at the 5'-end with LightCycler-Red 640, and to avoid extension modified at the 3'-end by phosphorylation. The other probe is labeled at 3'-end with fluorescein. Only after hybridization the template DNA, the two probes come in close proximity, resulting in fluorescent resonance energy transfer (FRET) between the two fluorophores. During FRET, fluorescein, the donor fluorophore, is excited by the light source of the LightCycler Instrument, and part of excitation energy is transferred to LightCycler-Red 640, the acceptor fluorophore. The LightCycler Instrument then measures the emitted fluorescence of LightCycler-Red 640. T. gondii repetitive DNA sequence is amplified with specific primers.  RT-PCR protocols, with the cycles (Taq Activation, Amplification and Melting curve and cooling) and the steps, were employed as described by manifacturer.  The limit of detection (LOD) is 10 copies/PCR reaction in clinical specimens. The amplicon is detected by fluorescence using a specific pair of Hybridization Probes labeled with Light Cycler-Red 640.
| Results|| |
In acute infection direct and indirect diagnostic techniques, used in this study, were always correlated. In particular in pregnant woman the pravelence was 5.2% (4/40), while in patients in suspicious of retinochoroiditis was 10% (1/10) [Table 1] and [Table 2]. In 3/4 of cases of amniotic fluid positive for DNA of T. gondii, the pregnant showed positive IgM and IgG, with low IgG avidity, therefore 1/4 of amniotic fluid positive for DNA of T. gondii showed only positive IgM [Table 1]. In the case of T. gondii ocular infection the RT-PCR and IgA were positive, whereas, the IgM and IgG were negative, as expected in a localized infection [Table 2]. [Table 3] and [Table 4] reveal no acute infection in all patients studied with serological and molecular assays.
|Table 1: Simultaneous use of diagnostic techniques in suspicion of infection from T. gondii in pregnant|
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|Table 2: Simultaneous use of diagnostic techniques in suspicion of retino-choroiditis from T. gondii|
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|Table 3: Simultaneous use of diagnostic techniques in suspicion of meningitis from T. gondii|
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|Table 4: Simultaneous use of diagnostic techniques in suspicion of blood infection from T. gondii|
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| Conclusions|| |
There are many difficulties in toxoplasmosis diagnosis, because it is an asymptomatic infection, causing significant morbidity and mortality, for this reason the "diagnostic-space" is very important.  Therefore a fast and accurate diagnosis of toxoplasmosis and the rapid treatment, for e.g., showed to be effective in reducing vertical trasmission severity. , Despite the limitations of the indirect diagnostic assays, ,,,,, the results obtained demonstrate that the serological techniques have a high diagnostic accuracy, given the high correlation with the results obtained with RT-PCR, as revealed for other pathogenic agents.  In conclusion, the use of molecular biology techniques is useful in pregnant women with recent seroconversion, to exclude the presence of T. gondii DNA in amniotic fluid. In addition, the RT-PCR, for detection T. gondii DNA, also appears an easy, accurate and rapid diagnostic technique, whose application is also useful in cases of borderline serologic results.
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[Table 1], [Table 2], [Table 3], [Table 4]