Development of monoclonal antibodies against Sj29 and its possible application for schistosomiasis diagnosis.

OBJECTIVE: Timely Schistosoma japonicum detection improves outcomes in schistosomiasis. Here, we established a double antibody sandwich ELISA to detect Schistosoma japonicum. METHODS: Sj29 polyclonal and monoclonal antibodies were developed and identified. A Sj29 double antibody sandwich ELISA was evaluated. RESULTS: Assay sensitivity for detecting Schistosoma japonicum circulating antigen Sj29 was 76.7% (23/30), 54.5% (18/33) and 50.0% (18/36) in patients with acute, chronic and advanced schistosomiasis. No false positives or cross-reactivity was observed in healthy controls or patients with clonorchiasis, paragonimiasis, or ancylostomiasis, respectively. By contrast, false positives (5.7%) and cross-reactivity (6.5%-10%) were detected using an AWA-ELISA. The circulating antigen positive rates decreased significantly faster than that of the antibody detection after 6 months treatment (22.2%, 4/18 and 88.9%, 16/18). Chi-Square Tests revealed that Sj29 sandwich ELISA had lower sensitivity than AWA indirect ELISA in the detection of S. japonicum infected patients (p<0.05). Although our assay detection specificity in patients infected with other parasites or healthy controls appeared higher, the difference between the assays was insignificant. However, our assay showed significantly better results in monitoring praziquantel therapeutic effects (p=0.001), with antigen-positive rates decreasing significantly faster than antibody detection rates after 6 months of treatment (22.2%, 4/18 versus 88.9%, 16/18). CONCLUSIONS: Sj29 double antibody sandwich ELISA was established. The specificity of this method for detecting healthy sera was 100%. Meanwhile, Sj29 sandwich ELISA may have a potential diagnostic capability to distinguish current from past infections and assess drug treatment responses.

Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples.

Loop-mediated isothermal amplification (LAMP), an attractive DNA amplification method, was developed as a valuable tool for the rapid detection of Toxoplasma gondii. In this study, species-specific LAMP primers were designed by targeting the AF146527 sequence, which was a conserved sequence of 200- to 300-fold repetitive 529 bp fragment of T.gondii. LAMP reaction system was optimized so that it could detect the minimal DNA sample such as a single tachyzoite or 10 copies of recombinant plasmid. No cross-reactivity was found when using DNA from other parasites as templates. Subsequently, a total of 200 human blood samples were directly investigated by two diagnostic methods, LAMP and conventional PCR. Fourteen of 200 (7%) samples were positive for Toxoplasma by LAMP (the primers developed in this study), whereas only 5 of 200 (2.5%) were proved positive by conventional PCR. The procedure of the LAMP assay was very simple, as the reaction would be carried out in a single tube under isothermal conditions at 64°C and the result would be read out with 1 h (as early as 35 min with loop primers). Thus, this method has the advantages of rapid amplification, simple operation, and easy detection and would be useful for rapid and reliable clinical diagnosis of acute toxoplasmosis, especially in developing countries.

[In vitro Effect of Hypericin against Toxoplasma gondii Tachyzoites].

Objective: To investigate the killing effect of hypericin on tachyzoites of Toxoplasma gondii RH strain in vitro. Methods: Normal saline （group A） and different concentrations of hypericin (5 µg/ml, group B; 50 µg/ml, group C; 500 µg/ml, group D） were added to T. gondii tachyzoites in 24-well plate（1×10(6)/well）. The tachyzoites were harvested after 2, 4 and 6 h, and underwent the following treatment: trypan blue staining to calculate the dyeing rate, Giemsa staining to observe the morphological and structural alterations of tachyzoites, and transmission electron microscopy to observe the ultrastructure of tachyzoites. In addition, flow cytometry was performed to calculate the survival rate of YFP-carrying Toxoplasma with the same treatment. Results: The trypan blue dyeing rate at 2 h after treatment in groups B, C and D was（11.0±3.6）%, （25.0±6.3）% and（40.0±2.7）% respectively, with a significant difference of group D versus B and C （P<0.01）, and groups C and D versus group A ［（6.0±3.0）%）］. The dyeing rate at 4 h and 6 h in group D was（97.0±2.0）% and （98.0±1.7）%, respectively, both significantly higher than that of groups C ［（30.0±7.2）%, （42.7±5.5）%ï¼½, B ［（20.0±3.0）%, （34.0±6.6）%ï¼½ and A ［（10.0±1.0）%, （19.3±4.9）%］（P<0.01）. Giemsa staining showed gradual end swelling and necrosis of tachyzoites with increased treatment duration and dosage. Transmission electron microscopy showed swelling of worm body, gap between cell membrane and matrix, increase and enlargement of vacuoles inside worm body, disruption of cell membrane, and dissolving of inner structures, with increased treatment duration. Flow cytometry showed significant difference of tachyzoite survival rate at 2, 4 and 6 h after hypericin treatment with that of the control group（P<0.01）. The survival rate of group C at 2 h after hypericin treatment was（7.9±1.9）%, significantly lower than that of groups B ［（38.1±5.5）%ï¼½ and A ［（81.8±6.0）%ï¼½ （P<0.01）. No tachyzoite was found to survive in group D at 2 h and in group C at 4 h. The survival rate of group B at 4 and 6 h after hypericin treatment was（14.3±7.9）% and （1.4±1.8）%, respectively, both significantly lower than that of group A［（73.8±11.3）% and（64.1±14.4）%, respectivelyï¼½ （P<0.01）. Conclusion: Hypericin has a remarkable killing effect on T. gondii tachyzoites, and the efficacy positively correlates with the dose and treatment duration.

Toxoplasma gondii: effects of exogenous nitric oxide on egress of tachyzoites from infected macrophages.

Toxoplasma gondii is an obligate intracellular parasite that can infect any nucleated cells of warm-blood vertebrates. Invasion and egress by this protozoan parasite, both of which are crucial for its life cycle, are rapid events that are dependent upon parasite motility. A variety of chemicals and molecules have been utilized to induce Toxoplasma early egress from host cells. Here, we aimed to determine whether nitric oxide (NO) could induce egress of T. gondii tachyzoites from infected cells. Infected macrophages were collected from C57BL/6 mice and treated with different doses of sodium nitroferricyanide (III) dihydrate (SNP) which releases nitric oxide into cell culture medium. The pattern of parasite egress was analyzed by flow cytometry. The results showed that exogenous NO released by SNP could trigger egress of T. gondii tachyzoites from infected peritoneal macrophages which then underwent necrosis after parasite egress. Our findings provided a novel approach to study the interactions between host immune responses and T. gondii.

Identification of antigenic proteins of Toxoplasma gondii RH strain recognized by human immunoglobulin G using immunoproteomics.

Toxoplasma gondii, a ubiquitous intracellular protozoan, infects one third of the world human population. It is of great medical significance, especially for pregnant women and immune-compromised patients. Accurate and early detection of T. gondii infection is crucial in the management of this disease. To obtain potential diagnostic markers, immunoproteomics was employed to identify immunodominant proteins separated by 2-D immunobloting and probed with sera collected from Toxoplasma-positive pregnant women. MALDI-TOF MS and MS/MS analyses identified a total of 18 immunoreactive proteins that were recognized by Toxoplasma-positive sera, whereas none was reactive with the negative-control sera from healthy, Toxoplasma-negative volunteers. Pregnant women showed a diverse immunoreactivity pattern with each serum recognizing one to eight identified tachyzoite proteins. The identified proteins were localized in the membrane, cytoplasm and specific organelles of T. gondii, and are involved in host cell invasion, metabolism and cell structure. Among these 18 proteins, actin, catalase, GAPDH, and three hypothetical proteins had a broad reactivity with Toxoplasma-positive sera, indicating their potential as diagnostic markers for toxoplasmosis. Each of several combinations of the identified proteins offered 100% detection of Toxoplasma infections of all 28 Toxoplasma-positive women. The study findings suggest that Toxoplasma tachyzoites are highly immunogenic and highlights the heterogeneity of host responses to Toxoplasma infection and the importance of using combinations of immunogens as diagnostic antigens. The findings have significant implications to the development of diagnostic reagents with high sensitivity and specificity.

Highly sensitive analysis of substituted aniline compounds in water samples by using oxidized multiwalled carbon nanotubes as an in-tube solid-phase microextraction medium.

An automated on-line method for the determination of the substituted aniline compounds was developed using in-tube solid-phase microextraction coupling to high-performance liquid chromatography (HPLC). In this work, oxidized multiwalled carbon nanotubes (MWCNTs-COOH) coated on the outer surface of the fused-silica tube and inserted in the polyether ether ketone (PEEK) tubing, which was fixed directly on the six-port injection valve to substitute for the sample loop. The extraction procedure was performed by a constant flow pump frequently driving the sample solution through the PEEK tubing and the analytes were adsorbed onto MWCNTs-COOH materials when the six-port valve set to load position. After extraction, the valve switched to inject position and the extracted analytes were desorbed by mobile phase in dynamic mode. High extraction capacity was achieved for the investigated analytes and great improvement of the limits of detection was obtained in comparison with other methods. The calibration plots were linear (r(2)> or =0.9949) over the concentration range of 1.04-104ngmL(-1) for 4-nitroaniline, 1.02-102ngmL(-1) for 2-nitroaniline, 1.68-168ngmL(-1) for 2-chloroaniline and 1.09-109ngmL(-1) for 2,4-dichloroaniline. The detection limit ranged from 0.04ngmL(-1) to 0.13ngmL(-1) (at S/N=3). The possibility of applying the established method to water samples analysis was also studied.