Evaluation of the siemens HIV antigen-antibody immunoassay.

Fourth-generation assays for the simultaneous detection of human immunodeficiency virus (HIV) antigen and antibodies are available on the international market and are currently used for blood donor screening and for HIV diagnosis. In this study we evaluated the performance of the novel automated fourth-generation ADVIA Centaur® HIV Ag/Ab Combo assay. The assay detected seroconversion at the same bleed or at least one bleed earlier in panels with respect to other assays and showed a detection efficacy equal to those of other assays in a low-titer panel. Samples obtained from blood donors (n = 2,778) or from HIV-positive patients (HIV-1 B subtype, n = 82; non-B subtype, n = 71) were also tested, showing a good correlation with other fourth-generation assays. We assessed the performance of 3 fourth-generation assays for detecting in utero transmitted anti-HIV antibodies and found a more specific detection efficiency with the ADVIA Centaur HIV Ag/Ab Combo assay compared to the other fourth-generation assays.

The second generation of HIV-1 vertically exposed infants: a case series from the Italian Register for paediatric HIV infection.

BACKGROUND: In the Highly Active Antiretroviral Therapy (HAART) era, the prognosis of children perinatally infected with HIV-1 has significantly improved, so the number of perinatally-infected females entering child-bearing age and experiencing motherhood is increasing. METHODS: A description of the medical history and pregnancy outcomes of women with perinatal acquired HIV-1 infection enrolled in the Italian Register for HIV infection in Children. RESULTS: Twenty-three women had 29 pregnancies. They had started an antiretroviral therapy at a median of 7.7 years (interquartile range, IQR 2.3 - 11.4), and had experienced a median of 4 therapeutic regimens (IQR 2-6). Twenty women (87%) had taken zidovudine (AZT) before pregnancy, in 14 cases as a starting monotherapy. In 21 pregnancies a protease inhibitor-based regimen was used. At delivery, the median of CD4+ T lymphocytes was 450/µL (IQR 275-522), and no viral load was detectable in 15 cases (reported in 21 pregnancies). Twenty-eight children were delivered through caesarean section (median gestational age: 38 weeks, IQR 36-38, median birth weight: 2550 grams, IQR 2270 - 3000). Intravenous AZT was administered during delivery in 26 cases. All children received oral AZT (median: 42 days, IQR 31 - 42), with no adverse events reported. No child acquired HIV-1 infection. CONCLUSIONS: Despite a long history of maternal infection, multiple antiretroviral regimens and, perhaps, the development of drug-resistant viruses, the risk of mother-to-child transmission does not seem to have increased among the second-generation of HIV-1 exposed infants.

Toxoplasma gondii Dense Granule Antigen 1 stimulates apoptosis of monocytes through autocrine TGF-ß signaling.

Monocyte/macrophages represent the first line of defense against protozoan parasites. Different mechanisms of monocyte suppression by Toxoplasma gondii that sustain parasite invasion and persistence have been described, including apoptosis. In the present study, we investigated the effect of microbial excretory­secretory polypeptides, namely the microneme protein MIC3 and the dense granule antigen GRA1, on apoptosis of monocytes from patients with congenital toxoplasmosis and healthy individuals. We found that GRA1 but not MIC3 could induce apoptosis of monocytes, observing the effect in samples from both Toxoplasma-infected and uninfected individuals, thus ruling out involvement of mechanisms of apoptosis linked to adaptive immunity or a cellular context related to infection. Selective inhibition of TGF-ß type I receptors reversed GRA1-induced apoptosis, indicating that this apoptosis involved canonical TGF-ß signaling. By using TGF-ß-neutralizing antibodies, we showed that monocyte apoptosis required endogenous TGF-ß and that GRA1 stimulation activated TGF-ß transcription and expression in monocytes but not lymphocytes, suggesting involvement of an autocrine TGF-ß-mediated mechanism in GRA1-induced apoptosis.

Prenatal treatment for serious neurological sequelae of congenital toxoplasmosis: an observational prospective cohort study.

BACKGROUND: The effectiveness of prenatal treatment to prevent serious neurological sequelae (SNSD) of congenital toxoplasmosis is not known. METHODS AND FINDINGS: Congenital toxoplasmosis was prospectively identified by universal prenatal or neonatal screening in 14 European centres and children were followed for a median of 4 years. We evaluated determinants of postnatal death or SNSD defined by one or more of functional neurological abnormalities, severe bilateral visual impairment, or pregnancy termination for confirmed congenital toxoplasmosis. Two-thirds of the cohort received prenatal treatment (189/293; 65%). 23/293 (8%) fetuses developed SNSD of which nine were pregnancy terminations. Prenatal treatment reduced the risk of SNSD. The odds ratio for prenatal treatment, adjusted for gestational age at maternal seroconversion, was 0.24 (95% Bayesian credible intervals 0.07-0.71). This effect was robust to most sensitivity analyses. The number of infected fetuses needed to be treated to prevent one case of SNSD was three (95% Bayesian credible intervals 2-15) after maternal seroconversion at 10 weeks, and 18 (9-75) at 30 weeks of gestation. Pyrimethamine-sulphonamide treatment did not reduce SNSD compared with spiramycin alone (adjusted odds ratio 0.78, 0.21-2.95). The proportion of live-born infants with intracranial lesions detected postnatally who developed SNSD was 31.0% (17.0%-38.1%). CONCLUSION: The finding that prenatal treatment reduced the risk of SNSD in infected fetuses should be interpreted with caution because of the low number of SNSD cases and uncertainty about the timing of maternal seroconversion. As these are observational data, policy decisions about screening require further evidence from a randomized trial of prenatal screening and from cost-effectiveness analyses that take into account the incidence and prevalence of maternal infection. Please see later in the article for the Editors' Summary.

Is the interruption of antiretroviral treatment during pregnancy an additional major risk factor for mother-to-child transmission of HIV type 1?

BACKGROUND: There is currently an experts' agreement discouraging interruption of antiretroviral treatment (ART) during the first trimester of pregnancy in women infected with human immunodeficiency virus type 1 (HIV-1). However, this recommendation is poorly supported by data. We evaluated the effects of discontinuing ART during pregnancy on the rate of mother-to-child transmission. METHODS: Logistic regression models were performed in a prospective cohort of 937 children who were perinatally exposed to HIV-1 to estimate adjusted odds ratios for confounding factors on mother-to-child transmission, including maternal interruption of ART. RESULTS: Among 937 pregnant women infected with HIV-1, ART was interrupted in 81 (8.6%) in the first trimester and in 11 (1.2%) in the third trimester. In the first trimester, the median time at suspension of ART was 6 weeks (interquartile range [IQR], 5-6 weeks) and the time without treatment was 8 weeks (IQR, 7-11 weeks). In the third trimester, the median time at suspension of ART was 32 weeks (IQR, 23-36 weeks) and the time without treatment was 6 weeks (IQR, 2-9 weeks). The plasma viral load was similar in women who had treatment interrupted in the first trimester and in those who did not have treatment interrupted. Overall, the rate of mother-to-child transmission in the whole cohort was 1.3% (95% confidence interval [CI], 0.7%-2.3%), whereas it was 4.9% (95% CI, 1.9%-13.2%) when ART was interrupted in the first trimester and 18.2% (95% CI, 4.5%-72.7%) when ART was interrupted in the third trimester. In the multiple logistic regression models, only interruption of ART during either the first or the third trimester, maternal mono- or double therapy, delivery by a mode other than elective cesarean delivery, and a viral load at delivery >4.78 log(10) copies/mL were independently associated with an increased rate of mother-to-child transmission. CONCLUSIONS: Discontinuing ART during pregnancy increases the rate of mother-to-child transmission of HIV-1, either when ART is stopped in the first trimester and subsequently restarted or when it is interrupted in the third trimester. This finding supports recommendations to continue ART in pregnant women who are already receiving treatment for their health.

Toxoplasma gondii in edible fishes captured in the Mediterranean basin.

The issue of whether market fish can be involved in the transmission of Toxoplasma gondii in the marine environment is highly debated since toxoplasmosis has been diagnosed frequently in cetaceans stranded along the Mediterranean coastlines in recent times. To support the hypothesis that fishes can harbour and effectively transmit the parasite to top-of-the-food-chain marine organisms and to human consumers of fishery products, a total of 1,293 fishes from 17 species obtained from wholesale and local fish markets were examined for T. gondii DNA. Real-time PCR was performed in samples obtained by separately pooling intestines, gills and skin/muscles collected from each fish species. Thirty-two out of 147 pooled samples from 12 different fish species were found contaminated with T. gondii DNA that was detected in 16 samples of skin/muscle and in 11 samples of both intestine and gills. Quantitative analysis of amplified DNA performed by both real-time PCR and digital PCR (dPCR) confirmed that positive fish samples were contaminated with Toxoplasma genomic DNA to an extent of 6.10 × 10-2 to 2.77 × 104  copies/ml (quantitative PCR) and of 1 to 5.7 × 104  copies/ml (dPCR). Fishes are not considered competent biological hosts for T. gondii; nonetheless, they can be contaminated with T. gondii oocysts flowing via freshwater run-offs (untreated sewage discharges, soil flooding) into the marine environment, thus acting as mechanical carriers. Although the detection of viable and infective T. gondii oocysts was not the objective of this investigation, the results here reported suggest that fish species sold for human consumption can be accidentally involved in the transmission route of the parasite in the marine environment and that the risk of foodborne transmission of toxoplasmosis to fish consumers should be further investigated.

Epidemiological and clinical features of pregnant women with HIV: a 21-year perspective from a highly specialized regional center in southern Italy.

PURPOSE: To analyze the changes over two decades in HIV-infected pregnant women followed at a highly specialized regional center for antenatal care in southern Italy. METHOD: Since 1985, all HIV-infected pregnant women attending our center have been monitored using progressively updated protocols. RESULTS: By December 2006, 230 deliveries in 159 women had been monitored. Deliveries in HIV-infected women increased from 0.16% (4/2,499) of all deliveries in 1985 to 0.73% (15/2,042) in 2006. The sociodemographic profile of the women changed greatly over the study period, and there was a shift from injecting drug use to heterosexual contact as the main transmission route and an increased proportion of foreign women. Subsequent to improvements in clinical care, the proportion of infected pregnant women receiving antiretroviral treatment increased from 27% (17/63) before 1996 to 81% (63/78) in 2006, with a corresponding decrease in the mother-to-child transmission rate from 36% (16/44) to 0.6% (1/157). CONCLUSION: The increasing number of HIV-infected pregnant women can be attributed to nonselective antenatal HIV screening, the spread of HIV infection through heterosexual contacts, and the desire of HIV-infected women to have children. In this context, highly specialized reference centers can play an important role in providing HIV-infected pregnant women with optimal care and in reducing mother-to-child transmission rates to very low levels.

Molecular dissection of the human B cell response against cytomegalovirus infection by lambda display.

Human cytomegalovirus (HCMV), a ubiquitous herpesvirus, is the main cause of congenital abnormalities and mental retardation in newborns and is also responsible for severe life-threatening complications in immunocompromised individuals, including AIDS patients and transplant recipients. The disorders generated by cytomegalovirus are closely associated with the competence of the host immune system and both humoral and cell-mediated mechanisms are involved in the response to viral infection. To identify viral proteins recognized by host antibody responses, a cytomegalovirus genome library was created and displayed on lambda bacteriophage. The challenge of such a library with sera from individuals with congenital or acquired infection allowed the identification of a wide panel of recombinant bacteriophages carrying cytomegalovirus B cell epitopes. Epitope-containing fragments within the families of tegument proteins (pUL25, pUL32), structural proteins (pUL48, pUL56) and glycoproteins (pUL55) were identified. Moreover, library screening permitted isolation of phage clones carrying an antigenic region of an uncharacterized HCMV protein encoded by the UL71 open reading frame (ORF), highlighting the potential of lambda display technology in antigen and epitope discovery.

Strategies for Prevention of Mother-to-Child Transmission Adopted in the "Real-World" Setting: Data From the Italian Register for HIV-1 Infection in Children.

BACKGROUND: Strategies for prevention of HIV-1 mother-to-child transmission (PMTCT) have been continuously optimized. However, cases of vertical transmission continue to occur in high-income countries. OBJECTIVES: To investigate changes in PMTCT strategies adopted by Italian clinicians over time and to evaluate risk factors for transmission. METHODS: Data from mother-child pairs prospectively collected by the Italian Register, born in Italy in 1996-2016, were analyzed. Risk factors for MTCT were explored by logistic regression analyses. RESULTS: Six thousand five hundred three children (348 infections) were included. In our cohort, the proportion of children born to foreign mothers increased from 18.3% (563/3078) in 1996%-2003% to 66.2% (559/857) in 2011-2016 (P < 0.0001). Combination neonatal prophylaxis use significantly (P < 0.0001) increased over time, reaching 6.3% (56/857) after 2010, and it was largely (4.2%) adopted in early preterm infants. The proportion of vaginal deliveries in women with undetectable viral load (VL) increased over time and was 9.9% (85/857) in 2011-2016; no infection occurred among them. In children followed up since birth MTCT, rate was 3.5% (96/2783) in 1996-2003; 1.4% (36/2480) in 2004-2010; and 1.1% (9/835) in 2011-2016. At a multivariate analysis, factors associated with MTCT were vaginal delivery with detectable or missing VL or nonelective caesarean delivery, prematurity, breastfeeding, lack of maternal or neonatal antiretroviral therapy, detectable maternal VL, and age at first observation. Previously described increased risk of offspring of immigrant women was not confirmed. CONCLUSIONS: Risk of MTCT in Italy is ongoing, even in recent years, underling the need for implementation of the current screening program in pregnancy. Large combination neonatal prophylaxis use in preterm infants was observed, even if data on safety and efficacy in prematures are poor.

Age-dependent impairment of functional helper T cell responses to immunodominant epitopes of Toxoplasma gondii antigens in congenitally infected individuals.

Human infection with Toxoplasma gondii is generally asymptomatic in immunocompetent adults while it causes significant morbidity in congenitally infected children. Cell mediated immunity plays the main role in host resistance to T. gondii infection and a Th1 cytokine profile is necessary for protection and control of infection. The present work focused on comparing the helper T cell response to the GRA1 antigen of the parasite between children with congenital toxoplasmosis and healthy adults with acquired infection. We demonstrated that in young children with congenital infection the specific T cell response to parasite antigens is impaired and that such hypo-responsiveness is restored during childhood. Also, we provided clear evidence that in individuals with congenital toxoplasmosis the acquisition of functional helper T cell responses is disease-unrelated and indistinguishable in terms of strength, epitope specificity, and cytokine profile from the corresponding responses in immunocompetent adults with asymptomatic acquired T. gondii infection.

Increased CD154 expression in uninfected infants born to HIV-positive mothers exposed to antiretroviral prophylaxis.

Although prevention of mother-to-child HIV transmission by antiretroviral drugs has been shown to be effective, the short- and long-term effects of treatment are not well known. Several reports suggest that antiretroviral drugs act not only by inhibiting viral replication, but also by improving antiviral immunity. In particular, treatment with nucleoside analogs was found to increase CD40 ligand (CD40L; CD154) levels, an inducible molecule expressed on activated T lymphocytes. The present study investigated potential immunostimulatory effects of antiretroviral treatment (ART) in uninfected HIV-exposed infants, receiving either ante- or postnatal therapy to reduce the transmission rate. To this end, we analyzed CD40L expression in unstimulated lymphocytes from peripheral blood samples of uninfected infants vertically exposed to HIV and subjected to ART. The CD45 PanLeucogating strategy was applied in flow cytometry to analyze the lymphocytes of 41 cases and 64 age-matched infants, taken as control. We found increased CD154 expression on both CD4+ and CD8+ lymphocytes in ART-treated infants, compared with the controls. CD154 was apparently functional, because the expression of CD86 on monocytes was enhanced; moreover, inhibition of the CD40-CD40L interaction produced downmodulation of CD86. From these results, we conclude that CD4+ and CD8+ lymphocytes of HIV-exposed noninfected infants, who have been exposed to antiretroviral drugs in fetal and early life, display enhanced CD154 expression and costimulatory activity.

Association between congenital toxoplasmosis and parent-reported developmental outcomes, concerns, and impairments, in 3 year old children.

BACKGROUND: Information is lacking on the effects of congenital toxoplasmosis on development, behavior, and impairment in later childhood, as well as on parental concerns and anxiety. This information is important for counselling parents about the prognosis for an infected child and for policy decisions on screening. METHODS: We prospectively studied a cohort of children identified by screening for toxoplasmosis in pregnant women or neonates between 1996 and 2000 in ten European centers. At 3 years of age, parents of children with and without congenital toxoplasmosis were surveyed about their child's development, behavior, and impairment, and about parental concerns and anxiety, using a postal questionnaire. RESULTS: Parents of 178/223 (80%) infected, and 527/821 (64%) uninfected children responded. We found no evidence that impaired development or behavior were more common in infected children, or that any potential effect of congenital toxoplasmosis was masked by prenatal treatment. Parents of infected children were significantly more anxious and reported more visual problems in their children. CONCLUSION: On average, children aged three to four years with congenital toxoplasmosis identified by screening and treated during infancy in this European setting had risks of abnormal development and behavior similar to uninfected children. Parental anxiety about infected children needs to be addressed by clinicians. Future studies with longer follow up and clinician-administered assessments may be better able to detect any subtle differences in child outcomes.

The Toxoplasma gondii bradyzoite antigens BAG1 and MAG1 induce early humoral and cell-mediated immune responses upon human infection.

Infection of humans by Toxoplasma gondii leads to an acute systemic phase, in which tachyzoites disseminate throughout the body, followed by a chronic phase characterized by the presence of tissue cysts, containing bradyzoites, in brain, heart and skeletal muscles. This work focused on studying the antigenic regions of bradyzoite-specific proteins involved in human B- and T-cell responses. To this aim, we constructed a phage-display library of DNA fragments derived from the bradyzoite-specific genes BAG1, MAG1, SAG2D, SAG4, BSR4, LDH2, ENO1 and p-ATPase. Challenge of the bradyzoite library with sera of infected individuals led to the identification of antigenic regions within BAG1 and MAG1 gene products. Analysis of the humoral and lymphoproliferative responses to recombinant antigens demonstrated that the BAG1 fragment induced T-cell proliferation in 34% of T. gondii-exposed individuals, while 50% of them had specific IgG. In the same subjects, the MAG1 fragment was recognized by T cells from 17% of the exposed donors and by antibodies from 73% of them. A detailed analysis of the antibody response against BAG1 and MAG1 antigen fragments demonstrated that the immune response against bradyzoites occurs early after infection in humans. Finally, we provide evidence that the T-cell response against BAG1 is associated with the production of interferon-gamma, suggesting that bradyzoite antigens should be considered in the design of potential vaccines in humans.

Molecular dissection of the human B-cell response against Toxoplasma gondii infection by lambda display of cDNA libraries.

The disorders generated by Toxoplasma gondii infection are closely associated with the competence of the host immune system and both humoral and cell mediated immunity are involved in response to parasite invasion. To identify antigens implicated in human B-cell responses, we screened a phage-display library of T. gondii cDNA fragments with sera of infected individuals. This approach identified a panel of recombinant phage clones carrying B-cell epitopes. All the peptide sequences selected by this procedure are regions of T. gondii gene products. These regions contain epitopes of the T. gondii antigens SAG1, GRA1, GRA7, GRA8 and MIC5, which are recognised by human immunoglobulins. Moreover, we report the isolation and characterisation of two additional immunodominant regions encoded by GRA3 and MIC3 genes, whose products have never been described as antigens of the human B-cell response against T. gondii infection. These results demonstrate potential of lambda-display technology for antigen discovery and for the study of the human antibody response against infectious agents.

Secular trend on congenital infections: insights from Campania region register for perinatal infection, southern Italy.

There is increasing pressure for prevention of vertically transmitted congenital infection and prenatal screening is recommended for main locally prevalent infections. Furthermore, systematic monitoring of their impact and effectiveness of practices is quite rare at National level. As a consequence of favorable conditions on Campania region of Southern Italy a monitoring system based on post- delivery surveillance on congenital infection has been longtime piloted and seems ready to be exported at national level. Temporal trend analysis is showing change on focus and impact of prevalent congenital infection such as toxoplasmosis, rubella, syphilis and HIV. Moreover, the effect of recent massive immigration from Eastern EU and Africa requires further focus.

Predictors of retinochoroiditis in children with congenital toxoplasmosis: European, prospective cohort study.

OBJECTIVE: By school age, 20% of children infected with congenital toxoplasmosis will have > or = 1 retinochoroidal lesion. We determined which children are most at risk and whether prenatal treatment reduces the risk of retinochoroiditis to help clinicians decide about treatment and follow-up. PATIENTS AND METHODS: We prospectively studied a cohort of children with congenital toxoplasmosis identified by prenatal or neonatal screening in 6 European countries. We determined the effects of prenatal treatment and prognostic markers soon after birth on the age at first detection of retinochoroiditis. RESULTS: Of 281 children with congenital toxoplasmosis, 50 developed ocular disease, and 17 had recurrent retinochoroiditis during a median follow-up of 4.1 years. Prenatal treatment had no significant effect on the age at first or subsequent lesions. Delayed start of postnatal treatment did not increase retinochoroiditis, but the analysis lacked power. Older gestational age at maternal seroconversion was weakly associated with a reduced risk of retinochoroiditis. The presence of nonocular clinical manifestations of congenital toxoplasmosis at birth strongly predicted retinochoroiditis. For 92% (230 of 249) of children with no retinochoroiditis detected before 4 months of age, the probability of retinochoroiditis by 4 years was low, whether clinical manifestations were present or not 8.0%. CONCLUSIONS: Prenatal treatment did not significantly reduce the risk of retinochoroiditis in this European cohort. If children have no retinochoroiditis in early infancy, the low risk of subsequent ocular disease may not justify postnatal treatment and repeated ophthalmic assessments during childhood. Controlled trials are needed to address the lack of evidence for the effectiveness of postnatal treatment.

Ocular sequelae of congenital toxoplasmosis in Brazil compared with Europe.

BACKGROUND: Toxoplasmic retinochoroiditis appears to be more severe in Brazil, where it is a leading cause of blindness, than in Europe, but direct comparisons are lacking. Evidence is accumulating that more virulent genotypes of Toxoplasma gondii predominate in South America. METHODS: We compared prospective cohorts of children with congenital toxoplasmosis identified by universal neonatal screening in Brazil and neonatal or prenatal screening in Europe between 1992 and 2003, using the same protocol in both continents. RESULTS: Three hundred and eleven (311) children had congenital toxoplasmosis: 30 in Brazil and 281 in Europe, where 71 were identified by neonatal screening. Median follow up was 4.1 years in Europe and 3.7 years in Brazil. Relatively more children had retinochoroiditis during the first year in Brazil than in Europe (15/30; 50% versus 29/281; 10%) and the risk of lesions by 4 years of age was much higher: the hazard ratio for Brazil versus Europe was 5.36 (95%CI: 3.17, 9.08). Children in Brazil had larger lesions, which were more likely to be multiple and to affect the posterior pole (p<0.0001). In Brazil, visual impairment (<6/12 Snellen) was predicted for most affected eyes (87%, 27/31), but not in Europe (29%; 20/69, p<0.0001). The size of newly detected lesions decreased with age (p = 0.0007). CONCLUSIONS: T. gondii causes more severe ocular disease in congenitally infected children in Brazil compared with Europe. The marked differences in the frequency, size and multiplicity of retinochoroidal lesions may be due to infection with more virulent genotypes of the parasite that predominate in Brazil but are rarely found in Europe.

Genetic and epigenetic factors at COL2A1 and ABCA4 influence clinical outcome in congenital toxoplasmosis.

BACKGROUND: Primary Toxoplasma gondii infection during pregnancy can be transmitted to the fetus. At birth, infected infants may have intracranial calcification, hydrocephalus, and retinochoroiditis, and new ocular lesions can occur at any age after birth. Not all children who acquire infection in utero develop these clinical signs of disease. Whilst severity of disease is influenced by trimester in which infection is acquired by the mother, other factors including genetic predisposition may contribute. METHODS AND FINDINGS: In 457 mother-child pairs from Europe, and 149 child/parent trios from North America, we show that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4. Polymorphisms at COL2A1 encoding type II collagen associate only with ocular disease. Both loci showed unusual inheritance patterns for the disease allele when comparing outcomes in heterozygous affected children with outcomes in affected children of heterozygous mothers. Modeling suggested either an effect of mother's genotype, or parent-of-origin effects. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting. CONCLUSIONS: These associations between clinical outcomes of congenital toxoplasmosis and polymorphisms at ABCA4 and COL2A1 provide novel insight into the molecular pathways that can be affected by congenital infection with this parasite.

Chimeric antigens of Toxoplasma gondii: toward standardization of toxoplasmosis serodiagnosis using recombinant products.

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.