Use of Recombinant Antigens for Early Postnatal Diagnosis of Congenital Toxoplasmosis

Buffolano, Wilma; Beghetto, Elisa; Pezzo, Mariassunta Del; Spadoni, Andrea; Cristina, Manlio Di; Petersen, Eskild; Gargano, Nicola

doi:10.1128/jcm.43.12.5916-5924.2005

Cited by 46 publications

(35 citation statements)

References 44 publications

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“…Several other recombinant antigens give similar results, but they were not included in the commercial assays. As the results were similar but weaker than for whole extract ELISA, attempts have been made to introduce a mixture of several recombinant antigens or chimeric proteins composed of several epitopes of diverse T. gondii proteins (BUFFOLANO et al, 2005;BEGHETTO et al, 2006;PIETKIEWICZ et al, 2007). However, all attempts have given similar results without improving upon the results of the whole extract ELISA.…”

Section: Discussionmentioning

confidence: 99%

“…There are several strains of T. gondii with variable virulence and some antigenic differences, despite the fact that all are reactive to whole extract antigens (KONG et al, 2003). These minor antigenic differences could affect ELISA with few epitopes, as observed in most isolated recombinant protein ELISA, which usually present less sensitivity and specificity than whole extract ELISA (BUFFOLANO et al, 2005). Another aspect is the parasite load in human infection, which can be acquired by oocyst ingestion of only eight sporozoites, or by tissue cysts in raw meat, containing several cysts with thousands of bradyzoites, as reported in experimental models comparing these two ways of infection and reporting that cyst induced infection was more acute and severe than oocyst induced infection (DUBEY, 2006).…”

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confidence: 99%

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Serology using rROP2 antigen in the diagnostic of toxoplasmosis in pregnant women

Macre¹,

Pires²,

Meireles³

et al. 2009

Rev. Inst. Med. trop. S. Paulo

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SUMMARYToxoplasma gondii causes severe fetal disease during acute infection in pregnant women, thus demanding early diagnosis for effective treatment and fetus preservation. Fetal tests are inefficient and risky, and diagnosis is based on maternal IgM serology, which had weak screening ability due to increased sensitivity, with alternative IgG avidity tests. Here, we performed ELISA and avidity assays using a recombinant T. gondii antigen, rROP2, in samples from 160 pregnant women screened from a large public hospital who were referred due to positive IgM assays. IgG serology and avidity assays were compared using whole T. gondii extract or rROP2. ELISA IgG detection with rROP2 showed good agreement with assays performed with T. gondii extract, but rROP2 IgG avidity assays were unrelated to whole extract antigen IgG avidity, regardless of the chaotrope used. These data show that avidity maturation is specific to individual antigen prevalence and immune response during infection. ELISA rROP2 IgG assays may be an alternative serological test for the diagnosis of toxoplasmosis during pregnancy, although our data do not support their use in avidity assays.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Serology using rROP2 antigen in the diagnostic of toxoplasmosis in pregnant women

Macre¹,

Pires²,

Meireles³

et al. 2009

Rev. Inst. Med. trop. S. Paulo

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“…Under these proteins, classic secretory antigens like macrophage-inhibitory cytokine 2 (MIC2), MIC5 (H4), GRA1, and GRA7 were observed. Judging as a proof of principle, these proteins have already been previously described as diagnostic markers (4,16,24). Besides, a protein with a molecular mass of about 80 kDa and a pI of 5 reacted also with Toxoplasma-specific IgA antibodies of patients with an acute infection.…”

Section: Identification Of Sub1 By 2-de and Mass Spectrometry Analysismentioning

confidence: 99%

Identification of Toxoplasma gondii SUB1 Antigen as a Marker for Acute Infection by Use of an Innovative Evaluation Method

Hruzik¹,

Asif²,

Groß³

2011

J Clin Microbiol

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By the separation of Toxoplasma lysate using two-dimensional gel electrophoresis and its analysis with human serum samples and mass spectrometry, the subtilisin-like protein (SUB1) was identified to be a potential marker for acute toxoplasmosis. Following expression of the C-terminal domain of SUB1 in Escherichia coli, it was tested in a line blot assay using a total of 80 human serum samples. Two computer programs based on different evaluation strategies were used for judgment of the line blot results: (i) a time-dependent method with a predefined cutoff value and (ii) a fixed-time-point method with a calculated cutoff. Thereby, SUB1 was proven to be rather reactive with specific immunoglobulin A (IgA), IgM, and IgG of patients with an acute infection. This finding makes this antigen an attractive candidate for improving diagnosis of toxoplasmosis and demonstrates that not only the selection of respective antigens but also the evaluation method chosen are important for the evaluation of new diagnostic markers.One of the most successful protozoan parasites is Toxoplasma gondii. In humans and other intermediate hosts, the tachyzoite stage of the parasite represents the active phase of infection, whereas the bradyzoite stage represents the chronic or latent phase of infection. In immunocompetent human hosts, the infection is asymptomatic in most cases. A special threat exists when primary infection takes place in the developing fetus or in immunocompromised hosts, particularly patients suffering from AIDS (21, 27) and transplant recipients (6, 27).Many commercial tests are available for the serological diagnosis of T. gondii infection. Here, the enzyme-linked immunosorbent assay (ELISA) and immunoblotting are widespread techniques that are performed automatically or manually for the detection of T. gondii-specific immunoglobulin G (IgG), IgM, and IgA.We tried to identify Toxoplasma antigens that might be suitable for the serological identification of early infection by twodimensional (2D) gel electrophoresis and mass spectrometry (MS) analysis. After recombinant production of the IgA-reactive T. gondii protein subtilisin-like protein 1 (SUB1), we evaluated it on human serum samples which were previously tested with commercial tests and classified as having either no T. gondii infection, acute infection (high IgM or IgA titer), or chronic infection (no IgM or IgA, high IgG titer). MATERIALS AND METHODSParasites and host cells. Tachyzoites of the type II strain NTE were isolated from an L929 fibroblast-T. gondii coculture. These parasites were used to infect human foreskin fibroblasts (HFFs) and cultured in Dulbecco modified Eagle medium containing 10% fetal calf serum, 1% penicillin-streptomycin, 1% sodium pyruvate, and 1% nonessential amino acids (NEAA) for 3 to 4 days. Infected cells were cultured at 37°C in 5% CO 2 atmosphere.T. gondii antigen preparation. Extracellular parasites were isolated from the supernatant of infected HFFs by filtration (pore size, 0.02 m; Millipore, Billerica, MA). The cell pellet of 4 ...

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“…Moreover, in 30% to 60% of infants with congenital toxoplasmosis, the Toxoplasma-specific IgM antibody response is absent or undetectable by standard serological assays (7,8,10,13). Therefore, the availability of innovative diagnostic methods would be desirable.During the last 10 years, several studies have reported on the use of recombinant antigens for the serological diagnosis of T. gondii infection (1,4,8,17,21,22,27,31). Nevertheless, although they are promising, none of the assays based on recombinant antigens displayed all the characteristics required to replace the tachyzoite antigen in IgG-and IgM-based tests, indicating that further work is needed before an immunoassay with recombinant products will be available for clinical purposes.…”

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confidence: 99%

“…However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins (18,23,24,34), due to the lack of a purified standardized Toxoplasma antigen or standard methods for preparation of the antigen. Moreover, in 30% to 60% of infants with congenital toxoplasmosis, the Toxoplasma-specific IgM antibody response is absent or undetectable by standard serological assays (7,8,10,13). Therefore, the availability of innovative diagnostic methods would be desirable.…”

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confidence: 99%

Chimeric Antigens ofToxoplasma gondii: Toward Standardization of Toxoplasmosis Serodiagnosis Using Recombinant Products

Beghetto¹,

Spadoni²,

Bruno³

et al. 2006

J Clin Microbiol

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We have evaluated the diagnostic utility of six antigenic regions of the Toxoplasma gondii MIC2, MIC3, M2AP, GRA3, GRA7, and SAG1 gene products, assembled in recombinant chimeric antigens by genetic engineering, in order to replace the soluble, whole-cell tachyzoite extract in serological assays. Serum samples from 100 adults with acquired T. gondii infection and from 30 infants born to mothers with primary toxoplasmosis contracted during pregnancy, of whom 20 were congenitally infected, were included. Immunoglobulin G (IgG) and IgM antibodies against epitopes carried by chimeric antigens were measured by performing parallel enzyme immunoassays (recombinant enzyme-linked immunosorbent assays [Rec-ELISAs]), and the results obtained by standard commercial assays with the whole-cell Toxoplasma antigen and assays with the chimeric antigens were compared. Our results demonstrate that IgG and IgM Rec-ELISAs with individual chimeric antigens have performance characteristics comparable to those of the corresponding commercial assays. Furthermore, we show that IgM-capture assays based on chimeric antigens improve the ability to diagnose congenital toxoplasmosis postnatally compared with the ability to diagnose congenital toxoplasmosis by the use of standard assays. The use of recombinant chimeric antigens is effective in distinguishing T. gondii-infected individuals from T. gondii-uninfected individuals and shows that immunoassays based on recombinant products could provide the basis for standardized commercial tests for the serodiagnosis of toxoplasmosis.Human infection by Toxoplasma gondii is generally asymptomatic and induces a self-limiting disease. In contrast, primary T. gondii infection acquired during gestation can be transmitted to the fetus through the placenta and may cause miscarriage, permanent neurological damage, premature birth, and visual impairment (15,29,32). Toxoplasmosis during gestation represents a formidable task for the clinician due to its subclinical course in the majority of pregnant women and the unpredictable long-term outcome of congenital infection (11,16,32). To implement suitable therapies in good time and to avoid neonatal malformations or reduced eyesight in newborns, it is essential to establish when the primary infection has been acquired in the mother and to determine if vertical transmission to the fetus has occurred.The diagnosis of T. gondii infection can be established by detecting parasite-specific DNA sequences in body fluids and tissues or Toxoplasma-specific immunoglobulins in sera from infected individuals (2,20,30). Serological methods are generally preferred, since the sensitivities and the specificities of other methods can be affected by the appropriateness of sample handling and shipping and storage conditions. Furthermore, parasitemia may be of short duration, further limiting the value of detection of DNA in amniotic fluid and peripheral blood.The diagnosis of toxoplasmosis by serological methods routinely uses immunoenzymatic assays to detect the presence of the var...

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Use of Recombinant Antigens for Early Postnatal Diagnosis of Congenital Toxoplasmosis

Cited by 46 publications

References 44 publications

Serology using rROP2 antigen in the diagnostic of toxoplasmosis in pregnant women

Serology using rROP2 antigen in the diagnostic of toxoplasmosis in pregnant women

Identification of Toxoplasma gondii SUB1 Antigen as a Marker for Acute Infection by Use of an Innovative Evaluation Method

Chimeric Antigens ofToxoplasma gondii: Toward Standardization of Toxoplasmosis Serodiagnosis Using Recombinant Products

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