Evaluation of a Recombinant Multiepitope Peptide for Serodiagnosis of Toxoplasma gondii Infection

Detection of Toxoplasma gondii infection with sensitive and specific methods is a key step in the prevention and treatment of toxoplasmosis. Among the available diagnostic tests, serology is commonly used. Although serological tests give satisfactory results, the production of reliable reagents remains laborious and expensive. There is therefore a real need to acquire specific and effective recombinant antigens for the serodiagnosis of T. gondii infection. In this study, a multiepitope peptide was designed and successfully expressed in Escherichia coli, and then IgG and IgM enzyme-linked immunosorbent assays (ELISAs) were developed and evaluated. Our results showed that the new multiepitope antigen is one of the most promising recombinant antigens which could be used in routine screening of human toxoplasmosis.     

Serodiagnosis of Recently Acquired Toxoplasma gondii Infection Using an Enzyme-Linked Immunosorbent Assay with a Combination of Recombinant Antigens

An enzyme-linked immunosorbent assay (ELISA) using four recombinant antigens of Toxoplasma gondii (rP22, rP25, rP29, and rP35) was used in an attempt to differentiate pregnant women with toxoplasma serologic profiles (TSPs) indicative of recently acquired infections (acute profile) from those with TSPs indicative of infections acquired in the distant past (chronic profile). In general, immunoglobulin G antibodies in sera from women with the acute profile reacted more strongly with the recombinant antigens than did those in sera from women with the chronic profile. However, reactivities differed significantly between antigens that reacted with a single serum and between sera that reacted with a single antigen. Because of these variations, we employed a combination of the four antigens in an ELISA (Comb-ELISA) and evaluated its ability to distinguish pregnant women with the acute profile from those with the chronic profile. Eighteen of 20 (90%) sera from acute-profile women were positive in the Comb-ELISA, whereas 69 of 70 (98.6%) sera from the chronic-profile women were negative. Thus, the Comb-ELISA may be useful for diagnosis of toxoplasmosis in pregnant women and for differentiation between recently acquired infections and infections acquired in the more distant past.     

A New MIC1-MAG1 Recombinant Chimeric Antigen Can Be Used Instead of the Toxoplasma gondii Lysate Antigen in Serodiagnosis of Human Toxoplasmosis

This study presents an evaluation of the MIC1 (microneme protein 1)-MAG1 (matrix antigen 1) Toxoplasma gondii recombinant chimeric antigen for the serodiagnosis of human toxoplasmosis for the first time. The recombinant MIC1-MAG1 antigen was obtained as a fusion protein containing His tags at the N- and C-terminal ends using an Escherichia coli expression system. After purification by metal affinity chromatography, the chimeric protein was tested for usefulness in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-T. gondii immunoglobulin G (IgG). One hundred ten sera from patients at different stages of infection and 40 sera from seronegative patients were examined. The results obtained for the MIC1-MAG1 chimeric antigen were compared with those of IgG ELISAs using a Toxoplasma lysate antigen (TLA), a combination of recombinant antigens (rMIC1ex2-rMAG1) and single recombinant proteins (rMIC1ex2 and rMAG1). The sensitivity of the IgG ELISA calculated from all of the positive serum samples was similar for the MIC1-MAG1 chimeric antigen (90.8%) and the TLA (91.8%), whereas the sensitivities of the other antigenic samples used were definitely lower, at 69.1% for the mixture of antigens, 75.5% for the rMIC1ex2, and 60% for rMAG1. This study demonstrates that the MIC1-MAG1 recombinant chimeric antigen can be used instead of the TLA in the serodiagnosis of human toxoplasmosis.     

Recombinant Leishmania Antigens for Serodiagnosis of Visceral Leishmaniasis

Serological tests with crude or recombinant Leishmania antigens are important tools for the diagnosis of leishmania infection. However, these tests are not markers of active visceral leishmaniasis (VL), since antibodies to these markers are often observed in individuals with subclinical L. chagasi infection and they do not fall shortly after therapy. In this study, levels of immunoglobulin G (IgG) against three recombinant Leishmania antigens (rH2A, KMP11, and the “Q” protein) were evaluated in sera from individuals with subclinical L. chagasi infection and in patients with VL pre- and posttherapy. The sensitivity of the serological test for diagnosis of VL was 100% with all three antigens. The titers of IgG fell significantly after therapy. While most of the individuals with subclinical L. chagasi infection had antibodies to rH2A and the “Q” protein, only 1 out of 15 individuals had antibodies to KMP11. These data indicate that KMP11 may be used to discriminate L. chagasi infection from active VL and may serve as a marker of response to therapy.     

Seroreactivity to and Avidity for Recombinant Antigens in Toxoplasmosis

To improve serodiagnostic methods for the diagnosis of acute toxoplasmosis during pregnancy, a new test system has been developed and evaluated based on the use of recombinant antigens. Five recombinant Toxoplasma gondii antigens (ROP1, MAG1, SAG1, GRA7, and GRA8) were cloned in Escherichia coli, purified, and applied directly onto nitrocellulose membranes in a line assay (recomLine Toxoplasma). A panel of 102 sera from 25 pregnant women with supposed recent toxoplasmosis and from two symptomatic children was compared to a panel of 71 sera from individuals with past infection. Both panels were analyzed using a recombinant line assay for immunoglobulin G (IgG), IgM, and IgA antibodies and a reference enzyme-linked immunosorbent assay. Within the IgM-positive samples, antibodies against ROP1 were predominant regardless of the infection state. In IgG analysis a characteristic antibody pattern was found for very recent infections. This pattern changed to a different one during the time course of infection: antibodies against GRA7 and GRA8 were characteristic for very early IgG, whereas antibodies against SAG1 and MAG1 appeared significantly later. These results were further confirmed by determination of the IgG antibody avidity for every single recombinant antigen. In the time course of infection, IgG antibodies against the early recognized antigens matured significantly earlier than those directed against the later antigens did. The IgA patterns did not give reliable information about the infection time points. The data revealed that the recombinant line assay provides valuable information on the actual state of infection, especially during the early infection time points.     

Cellular Immune Responses to Recombinant Antigens in Pregnant Women Chronically Infected with Toxoplasma gondii

The parasite Toxoplasma gondii can infect most mammals and birds, sometimes causing severe pathology. Primary infection during pregnancy can result in abortion or fetal defects. Host immunity, particularly cellular immunity towards antigenic peptides, can control infection, but an efficient vaccine is not yet available. We have evaluated T-cell responses to a crude soluble toxoplasma antigen (ST-Ag) and to five recombinant peptide antigens of cells in whole-blood cultures from 22 pregnant women with preexisting infections and from 7 pregnant negative controls. Cells from all infected patients but from none of the controls responded specifically to ST-Ag by expressing surface CD25 on culture. Responses to the recombinant antigens showed considerable variation between individuals. rGRA1 elicited a response in 16 of the 22 samples (73%), rSAG1 in 13, rGRA7 in 9, rGRA6-CT in 4, and rGRA6-NT in only 1. Most responding cells were CD4⁺. Cells from infected subjects cultured with ST-Ag all released high levels of gamma interferon (IFN-γ) into the culture supernatant (4,343 ± 2,536 pg/ml). Cells from 12 patients released IFN-γ after culture with rGRA1 (130 ± 98 pg/ml), those from 10 patients released it after culture with rSAG1 (183 ± 128 pg/ml), and those from 4 patients released it after culture with rGRA7 (324 ± 374 pg/ml). Intensity of IFN-γ production in response to the latter two recombinant antigens correlated with responses to ST-Ag (r = 0.61 and 0.53, respectively; P < 0.01). Interleukin-4 was always absent from supernatants of cells stimulated with toxoplasma antigens. The heterogeneity of human responses to individual recombinant toxoplasma antigens should be considered in the design of potential vaccines.     

Multicenter Evaluation of Strategies for Serodiagnosis of Primary Infection with Toxoplasma gondii

 The diagnostic performance of single-serum assays for toxoplasma-specific immunoglobulin (Ig)M, IgA, IgG, and IgE antibodies and of different combinations of such antibody assays in 20 European reference centers was assessed. A panel of 276 sera, of which 73 came from patients who seroconverted within 3 months (acute infection), 49 from patients who had seroconverted 3–12 months earlier (convalescence), and 154 from subjects who had two IgG-positive samples obtained 12 months apart (past infection), was tested with 20 toxoplasma-antibody assays and 195 combinations. In general, every assay with high diagnostic sensitivity showed low diagnostic specificity, i.e. no assay performed alone could reliably distinguish acute from past infection. Furthermore, no single assay (or combination) could separate convalescence from the other stages of toxoplasma infection. However, excellent diagnostic performances were reached by sequential use of highly sensitive IgM assays and methods examining IgG avidity or stage specificity. IgA or IgM assays were less suitable for confirmation of toxoplasma-IgM positivity. This study documents the strength of test combinations in assessing the stage of toxoplasma infection.     

Serodiagnosis of recently acquired Toxoplasma gondii infection with a recombinant antigen

A portion of a cDNA encoding a 35-kDa antigen from Toxoplasma gondii was cloned into the CKS expression vector and expressed in Escherichia coli. By using the enzyme-linked immunosorbent assay (ELISA), the recombinant protein (rP35 antigen) was examined for reactivity with immunoglobulin G (IgG) antibodies in the sera of pregnant women. Of these women, 41 had a toxoplasma serologic profile suggestive of recently acquired T. gondii infection (Sabin-Feldman dye test [DT] titers from 1:256 to 1:32,000, positive IgM ELISA titers from 2.3 to 9.7, positive IgA ELISA from 1 to >28, and acute patterns in the differential agglutination [AC/HS] test) (group I), and 50 women had a toxoplasma serologic profile suggestive of infection acquired in the distant past (low DT titers from 1:16 to 1:512, negative IgM ELISA titers from 0 to 0.8, and chronic patterns in the AC/HS test) (group II). The classification of acute or chronic profile was based on the individual's clinical history as well as the combination of the results of the toxoplasma serological profile. An additional group (group III) was composed of sera from 50 women who were seronegative for T. gondii antibodies in the DT. The results revealed that whereas 85.3% of women in group I had IgG antibodies that reacted with the rP35 antigen, only 8% of women in group II had IgG antibodies that reacted with the same antigen. In immunoblots, the rP35 antigen was recognized by IgG antibodies in a pool of sera from individuals with a toxoplasma serologic profile compatible with acute infection but not in a pool of sera from individuals with a serologic profile characteristic of a chronic infection. These results reveal that IgG antibodies against the P35 antigen are produced during the acute stage of the infection but are uncommon in the latent or chronic phase of the infection. Thus, the rP35 antigen may be a useful serologic marker to differentiate between recently acquired infection and that acquired in the more distant past.     

Human platelet inhibition of Toxoplasma gondii growth

The human platelet contribution against the intracellular growth of the parasite in vitro in human pulmonary fibroblasts was explored. It was observed that tachyzoites of Toxoplasma gondii induced activation of human platelets and additionally that platelets mediated inhibition of intracellular growth in a virulent T. gondii strain. A prominent role for platelet-derived growth factor (PDGF) was demonstrated in this phenomenon, by testing human recombinant PDGF-AA, -AB and -BB and antibodies to human PDGF-AB that partially reversed its effects. Moreover, the effect of PDGF was significantly higher if the host cells were treated 2 h before parasite infection. PDGF was not directly ‘toxic’ to free tachyzoites, but only affected parasites within host cells. PDGF-mediated inhibition may involve the cyclooxygenase cycle of the fibroblasts being partially reversed by the cyclooxygenase inhibitors, acetylsalicylic acid and indomethacin. However, a thromboxane synthetase pathway was not implicated. PDGF action against intracellular tachyzoites may also include increased IL-6 production in fibroblasts. Finally, transforming growth factor-beta 1 (TGF-β1), another component of α-granules released at the same time as PDGF, may not be antagonistic to the PDGF parasite inhibitory effect in confluent host cells.     

Recombinant Mouse-Human Chimeric Antibodies as Calibrators in Immunoassays That Measure Antibodies to Toxoplasma gondii

In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was ≥0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays.     

Recombinant Assay for Serodiagnosis of Lyme Disease Regardless of OspA Vaccination Status

All current seroassays using cultured Borrelia burgdorferi as their antigen source have been rendered obsolete by the recombinant OspA Lyme disease vaccine. OspA is the major outer surface protein expressed in cultured B. burgdorferi, and any seroassay that uses whole organisms as its antigen source cannot differentiate between subjects who received the vaccine and those who were naturally infected. We developed a new sensitive and specific enzyme-linked immunosorbent assay (ELISA) utilizing recombinant chimeric borrelia proteins devoid of OspA (rNon-OspA) that can be used to detect antibodies to diagnostically important B. burgdorferi antigens in both OspA-vaccinated and nonvaccinated individuals. We tested sera from patients with Lyme disease and with conditions associated with false-positive serologies, OspA-vaccinated individuals, and healthy high-risk workers from an area of endemicity and normal sera from individuals from areas of nonendemicity. The rNon-OspA test was compared with two commercially available whole-cell immunoassays. The rNon-OspA assay is as sensitive and specific as the whole-cell assay (P > 0.05) for detection of anti-B. burgdorferi antibodies. However, the rNon-OspA assay can differentiate between populations comprised of naturally infected and OspA-vaccinated individuals (P < 0.05). Our data demonstrate that this new sensitive rNon-OspA ELISA can be used for the laboratory detection of B. burgdorferi antibodies regardless of vaccination status and could replace existing serologic assays for Lyme disease.     

Enzyme-Linked Immunosorbent Assays Using the Recombinant Dense Granule Antigens GRA6 and GRA1 of Toxoplasma gondii for Detection of Immunoglobulin G Antibodies

The potential of the dense granule antigens GRA1 and GRA6 of Toxoplasma gondii to be used as diagnosis reagents in a recombinant form was evaluated. Both proteins were expressed in Escherichia coli as glutathione-S-transferase (GST) fusions. The GST-GRA1 fusion comprises the entire GRA1 sequence devoid of its N-terminal signal peptide. Separate expression of the two N- and C-terminal hydrophilic regions of GRA6 showed that only the N-terminal hydrophilic part of the protein was recognized by a pool of positive human sera in an immunoblot. One hundred T. gondii-positive and 98 negative human sera were tested in two separate immunoglobulin G (IgG)-direct enzyme-linked immunosorbent assays (ELISAs) using either GST-GRA1 or GST-GRA6-Nt recombinant protein. Whereas the sensitivity of the GST-GRA1 IgG ELISA was low (68%), the GST-GRA6-Nt IgG ELISA reached a sensitivity of 96%. The reactivity to GRA6-Nt was shown to be high even with human sera of low IgG titers. In addition, comparison of the optical density values for each serum revealed that GRA1 may complement GRA6-Nt to reach an overall sensitivity of 98%. Therefore, the GST-GRA6-Nt ELISA could be used together with another antigen like GRA1 for the development of a recombinant antigen-based test for serodiagnosis of toxoplasmosis.     

PCR-Based Detection of Toxoplasma gondii DNA in Blood and Ocular Samples for Diagnosis of Ocular Toxoplasmosis

PCR detection of Toxoplasma gondii in blood has been suggested as a possibly efficient method for the diagnosis of ocular toxoplasmosis (OT) and furthermore for genotyping the strain involved in the disease. To assess this hypothesis, we performed PCR with 121 peripheral blood samples from 104 patients showing clinical and/or biological evidence of ocular toxoplasmosis and from 284 (258 patients) controls. We tested 2 different extraction protocols, using either 200 μl (small volume) or 2 ml (large volume) of whole blood. Sensitivity was poor, i.e., 4.1% and 25% for the small- and large-volume extractions, respectively. In comparison, PCR with ocular samples yielded 35.9% sensitivity, while immunoblotting and calculation of the Goldmann-Witmer coefficient yielded 47.6% and 72.3% sensitivities, respectively. Performing these three methods together provided 89.4% sensitivity. Whatever the origin of the sample (ocular or blood), PCR provided higher sensitivity for immunocompromised patients than for their immunocompetent counterparts. Consequently, PCR detection of Toxoplasma gondii in blood samples cannot currently be considered a sufficient tool for the diagnosis of OT, and ocular sampling remains necessary for the biological diagnosis of OT.     

用重组SAG1抗原检测弓形虫抗体

目的探讨重组SAG1抗原对弓形虫IgG和IgM抗体的检测效果。方法用rSAG1作抗原建立免疫印迹方法（rSAG1-WB）,与玻片虫体过氧化物酶免疫染色试验（TSHE）平行检测不同来源血清。结果15例病原学检查阳性小鼠血清和5例免疫兔血清的IgG抗体均为阳性,30例正常小鼠血清和10例正常兔血清均未出现阳性反应。rSAG1-WB检测可疑弓形虫病患者血清阳性率为60．3％（38／63）,献血员血清阳性率为6％（3／50）,与TSHE检测结果（65．1％和4％）均无统计学差异（P〉0．05）。1例IgM强阳性血清和13例IgM弱阳性血清在Western—blot检测中分别出现相应的强阳性与弱阳性反应,50例献血员血清均未出现IgM阳性反应,结果与TSHE一致。结论rSAG1-WB检测弓形虫IgG和IgM抗体均具有高度的敏感性和特异性．与TSHE的符合率高。     

Antigens of Toxoplasma gondii with a diagnostic and potential immunoprophylactic value: new strategies of identification

Toxoplasma gondii is an ubiquitous protozoan parasite which induces severe pathology in in utero infected children and in immunosuppressed patients (particularly in the case of AIDS). Previous work that focused on toxoplasma somatic antigens failed to demonstrate an efficient protection against highly virulent T. gondii strains. The authors therefore first studied the role of parasite excreted-secreted (ES) antigens in the immune response. They describe here the preparation of excreted-secreted antigens in cell-free medium from tachyzoites, the intracellular proliferative stage present during acute infection. Major ES antigens have Mr of 108 K, 97 K, 86 K, 57 K, 42 K, 39 K, 28.5 K, 27 K and 21 K. The protective role of ES antigens has been demonstrated using congenitally athymic (Nu/Nu) rats that are highly sensitive to T. gondii infection (+/+ Fischer rats are resistant). The humoral and cellular components of this protection have been studied by the passive transfer either of sera or of T lymphocytes from ES-immunized +/+ Fischer rats into Nu/Nu rats. Adoptive transfers were carried out 24 hours before infection with the highly virulent T. gondii RH strain. Based on the concept of concomitant immunity, the authors have characterized antigens from tachyzoites and bradyzoites (the encysted stage persisting during chronic infection) which share common epitopes. Four tachyzoite antigens, P63, GP43, P39 and GP 28.5 have been shown by immunoprecipitation to cross-react with bradyzoite antigens. Two monoclonal antibodies raised against ES antigens permitted to demonstrate the localization of the 28.5 K and 27 K antigens inside the dense granules of tachyzoites and bradyzoites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and Biological and Molecular Characterization of Toxoplasma gondii from Canine Cutaneous Toxoplasmosis in Brazil

Cutaneous toxoplasmosis is a rare manifestation. This study represents a case report of an immunosuppressed dog that developed nodular dermal lesions caused by Toxoplasma gondii. The isolate (TgDgBr20) was characterized as mouse virulent and was genotyped as type BrI (ToxoDB genotype 6) using PCR-restriction fragment length polymorphism (RFLP) and as Africa 1 through microsatellite analysis.     

Detection of Human Toxoplasma-Specific Immunoglobulins A, M, and G with a Recombinant Toxoplasma gondii Rop2 Protein

The Toxoplasma gondii rhoptry protein Rop2 was expressed in Escherichia coli as a fusion protein containing 44 kDa of the 55-kDa mature Rop2, supplied with six histidyl residues at the N-terminal end (Rop2_196–561). Humoral response during Toxoplasma infection of humans was analyzed by immunoglobulin G (IgG), IgA, and IgM enzyme-linked immunosorbent assay with Rop2_196–561 as the antigen substrate. The analyzed sera were divided according to T. gondii-specific serological tests (IgG, IgA, or IgM indirect immunofluorescence and IgA or IgM immunosorbent agglutination assay) as group A (IgG⁺ IgA⁻ IgM⁻; n = 35), group B (IgG⁺ IgA⁺ IgM⁺; n = 21), group C (IgG⁺ IgA⁺ IgM⁻; n = 5), and group D (IgG⁺ IgA⁻ IgM⁺; n = 16). Twenty-six T. gondii-seronegative sera from individuals with other infections were also included (group E). Anti-Rop2 IgG antibodies were detected in 82.8% of group A sera and in 97.6% of the sera with acute-phase marker immunoglobulins (groups B, C, and D). The percentage of IgA antibody reactivity against Rop2_196–561 was 17.1% in group A, 50% in group D, and 80.8% in groups B and C. The percentage of IgM antibody reactivity was 0% in groups A and C and 62% in groups B and D. Sera from group E failed to show IgA, IgM, or IgG antibody reactivity. Since T. gondii Rop2 elicits a strong humoral response from an early stage of infection, it is suggested that recombinant Rop2_196–561 would be suitable for use in diagnostic systems, in combination with other T. gondii antigens, to detect specific IgG, IgA, and IgM antibodies.     

Performance of Purified Antigens for Serodiagnosis of Pulmonary Tuberculosis: a Meta-Analysis

Serological antibody detection tests for tuberculosis may offer the potential to improve diagnosis. Recent meta-analyses have shown that commercially available tests have variable accuracies and a limited clinical role. We reviewed the immunodiagnostic potential of antigens evaluated in research laboratories (in-house) for the serodiagnosis of pulmonary tuberculosis and conducted a meta-analysis to evaluate the performance of comparable antigens. Selection criteria included the participation of at least 25 pulmonary tuberculosis patients and the use of purified antigens. Studies evaluating 38 kDa, MPT51, malate synthase, culture filtrate protein 10, TbF6, antigen 85B, α-crystallin, 2,3-diacyltrehalose, 2,3,6-triacyltrehalose, 2,3,6,6′-tetraacyltrehalose 2′-sulfate, cord factor, and TbF6 plus DPEP (multiple antigen) were included in the meta-analysis. The results demonstrated that (i) in sputum smear-positive patients, sensitivities significantly ≥50% were provided for recombinant malate synthase (73%; 95% confidence interval [CI], 58 to 85) and TbF6 plus DPEP (75%; 95% CI, 50 to 91); (ii) protein antigens achieved high specificities; (iii) among the lipid antigens, cord factor had the best overall performance (sensitivity, 69% [95% CI, 28 to 94]; specificity, 91% [95% CI, 78 to 97]); (iv) compared with the sensitivities achieved with single antigens (median sensitivity, 53%; range, 2% to 100%), multiple antigens yielded higher sensitivities (median sensitivity, 76%; range, 16% to 96%); (v) in human immunodeficiency virus (HIV)-infected patients who are sputum smear positive, antibodies to several single and multiple antigens were detected; and (vi) data on seroreactivity to antigens in sputum smear-negative or pediatric patients were insufficient. Potential candidate antigens for an antibody detection test for pulmonary tuberculosis in HIV-infected and -uninfected patients have been identified, although no antigen achieves sufficient sensitivity to replace sputum smear microscopy. Combinations of select antigens provide higher sensitivities than single antigens. The use of a case-control design with healthy controls for the majority of studies was a limitation of the review. Efforts are needed to improve the methodological quality of tuberculosis diagnostic studies.The failure to diagnose tuberculosis (TB) accurately and rapidly is a key challenge in curbing the epidemic (45, 88, 116). Sputum microscopy, currently the sole diagnostic test in most areas where TB is endemic, has several limitations; in particular, the sensitivity compared with that of culture is variable (80, 97, 104, 116), multiple patient visits are required (56, 93, 114), considerable technical training is necessary, and the procedure is labor-intensive (45, 65). Antibody detection tests (serological tests) are used for the diagnosis of many infectious diseases and could potentially improve the means of diagnosis of TB. These tests measure the presence of specific antibodies (most often immunoglobulin G [IgG]) directed against immunodominant antigens of the pathogen in question. Compared with microscopy, antibody detection methods may enable the rapid diagnosis of TB, as these tests have the advantages of speed (results can be available within hours), technological simplicity, and minimal training requirements. In addition, these tests can be adapted to point-of-care formats that can be implemented at lower levels of health services in low- and middle-income countries (21, 22, 57, 65).Efforts to develop antibody detection tests for the diagnosis of TB have been under way for decades, and the performance of these tests has been well described (13, 17, 22, 32, 40, 47, 48, 52, 60, 64, 100, 107). Several systematic reviews of these tests have been published (discussed below) (28, 94, 95).First-generation antibody detection tests were based on crude mixtures of constituents and products of Mycobacterium tuberculosis, for example, culture filtrate proteins and purified protein derivative, the preparation used in the tuberculin skin test. Several of these early tests had low specificities, as the tests contained antigens shared among different bacterial species (1, 22, 48, 57). During the past two decades, an increased understanding of humoral immune responses to M. tuberculosis and the new tools of genomics and proteomics have led to the discovery of new antigens reported to provide improved sensitivities and specificities for the diagnosis of TB compared with those achieved with the antigens in the first-generation tests (48).We reviewed the immunodiagnostic potential of different antigens evaluated in research laboratories (in-house) for the serodiagnosis of pulmonary TB and carried out a meta-analysis to evaluate the performance of various antigens singly and in combination. Previous meta-analyses have shown that commercially available serological tests for both pulmonary TB (94) and extrapulmonary TB (95) have variable accuracies and, consequently, a limited clinical role. Another systematic review (searches through 2003) limited studies to the cohort or case series type of design and included only nine studies relating to in-house anti-TB antibody serological tests (28). A recently published expert review (1) did not include a meta-analysis. We are unaware of other systematic reviews on this topic.The current review addresses the following questions. (i) What is the performance of different antigens in the serodiagnosis of pulmonary TB in sputum smear-positive and smear-negative patients? (ii) What is the performance of these antigens in the serodiagnosis of pulmonary TB in patients with human immunodeficiency virus (HIV) infection?     

Use of Protein AG in an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Toxoplasma gondii Infection in Four Species of Animals

An enzyme-linked immunosorbent assay (ELISA) employing protein AG (AG-ELISA) as a conjugate was developed to detect anti-Toxoplasma gondii antibodies (Ab) in experimentally infected pigs and naturally infected pigs, goats, dogs, and cats. The results indicate that AG-ELISA can be a useful method for serological diagnosis of T. gondii infection in these four species of animals.Infections with Toxoplasma gondii are prevalent in human beings and animals worldwide (1). Numerous studies of T. gondii infections in pigs and other animals have demonstrated that enzyme-linked immunosorbent assay (ELISA) is the most sensitive method for diagnosing T. gondii infection (2, 4, 8). However, ELISA needs species-specific secondary antibodies for testing each species of animals. This requirement increases cost and makes manipulation more complicated when samples from different animal species are examined. Since the chimeric protein AG can strongly bind to the IgG of many mammalian species, it has been a powerful tool in immunological studies (5, 6). So far, there has been no report on the utility of chimeric AG in the immunological diagnosis of T. gondii infection. In this study, chimeric protein AG was employed to develop a simple protein AG enzyme-linked immunosorbent assay (AG-ELISA) to detect T. gondii antibodies from different animal species.For establishing the AG-ELISA method, recombinant MIC3, which was highly expressed in Escherichia coli by our laboratory as previously described (9), was used as the coating antigen. Flat-bottomed 96-well polystyrene micro-titration plates were coated with 0.1 ml of the antigens (2.5 mg/liter) diluted in 0.05 M carbonate buffer (pH 9.6) by incubation overnight at 4°C and were blocked with carbonate buffer-1% ovalbumin for 1 h at 37°C. The control and test sera were diluted 1:160 in phosphate-buffered saline-Tween (PBST) containing 0.1% ovalbumin, added to the microtiter plate at 0.1 ml per well, and incubated for 1 h at 37°C. Next, peroxidase-labeled chimeric protein AG (1:4,000; Pierce, Rockford, IL) was added at 0.1 ml per well and incubated for 30 min at 37°C. Peroxidase activity was revealed by adding 0.1 ml of tetramethylbenzidine (TMB) solution (100 mg TMB/liter of phosphate citrate buffer [pH 6.0] and 200 ul of H₂0₂) for 10 min at room temperature. The reaction was stopped by adding 0.05 ml of 0.25% hydrofluoric acid (HF), and the optical density (OD) was read at 630 nm in an ELISA microplate reader. A serum sample was considered to be positive when the OD of the sample/the OD of the negative control was ≥2.3.Twelve mixed-breed pigs between 6 and 8 weeks old were purchased from one pig farm and randomly allocated to separate stables. The animals were acclimatized for 7 days before use. All pigs tested negative for the presence of T. gondii antibodies by AG-ELISA (OD < 0.16) and modified agglutination test (MAT) (titer < 1:16). At day 0, eight pigs were inoculated with 2 × 10⁴ of viable tachyzoites of the RH strain by the subcutaneous route; the other four pigs were used as controls. Serum samples were collected from all groups, including the control group, on days −7, 0, 7, 10, 14, 21, 28, 35, 42, 50, and 57 after infection, and AG-ELISA was performed. MAT (3) and a validated commercial ELISA kit (SafePath Laboratories, Carlsbad, CA) were used as controls. The ELISA kit was able to detect that all animals were positive on day 7 after challenge, whereas AG-ELISA detected positive results on day 10 and MAT on day 14. The average antibody level was highest on day 28 for the ELISA kit and on day 35 for AG-ELISA and MAT. The reason for these differences may be that the ELISA kit uses formalin-fixed whole tachyzoites as the antigen and detects antibodies to surface antigen, whereas AG-ELISA uses MIC3 as the coating antigen and measures antibodies to secretory antigen. These results also indicate that ELISA methods are more sensitive than MAT. Agreement among these three serologic tests was calculated by Kappa statistics (7), and good agreement was observed with AG-ELISA versus MAT (κ = 0.86, P < 0.001) and AG-ELISA versus ELISA (κ = 0.83, P < 0.001).A total of 304 serum samples were collected from 2006 to 2008 at the clinic of the animal hospital of our university from four species of animals, including pigs (n = 197), goats (n = 60), dogs (n = 24), and cats (n = 13). All samples were tested by the AG-ELISA and MAT methods, and the results are shown in Table ​Table1.1. Although AG-ELISA showed higher prevalences than MAT for pigs (x² = 0.87, P > 0.05) and goats (x² = 0.36, P > 0.05), the difference was not statistically significant. Both methods detected the same prevalence for dogs and cats. Kappa results were assessed, and good agreement was observed for AG-ELISA versus MAT (κ = 0.85, P < 0.001). All these results indicate that AG-ELISA could be a useful method for serological detection of T. gondii infection in different species of animals. This is the first report on the utility of chimeric AG in the immunological diagnosis of T. gondii infection.

TABLE 1.

Comparison of numbers of Toxoplasma gondii-positive results and prevalences of T. gondii infection in clinical serum samples from pigs, goats, dogs, and cats determined by AG-ELISA and MAT

Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase.

Toxoplasma gondii lacks the capacity to synthesize purines de novo, and adenosine kinase (AK)-mediated phosphorylation of salvaged adenosine provides the major route of purine acquisition by this parasite. T. gondii AK thus represents a promising target for rational design of antiparasitic compounds. In order to further our understanding of this therapeutically relevant enzyme, an AK cDNA from T. gondii was overexpressed in E. coli using the pBAce expression system, and the recombinant protein was purified to apparent homogeneity using conventional protein purification techniques. Kinetic analysis of TgAK revealed Km values of 1.9 microM for adenosine and 54.4 microM for ATP, with a k(cat) of 26.1 min(-1). Other naturally occurring purine nucleosides, nucleobases, and ribose did not significantly inhibit adenosine phosphorylation, but inhibition was observed using certain purine nucleoside analogs. Adenine arabinoside (AraA), 4-nitrobenzylthioinosine (NBMPR), and 7-deazaadenosine (tubercidin) were all shown to be substrates of T. gondii AK. Transgenic AK knock-out parasites were resistant to these compounds in cell culture assays, consistent with their proposed action as subversive substrates in vivo.