Comparative evaluation of plant extract effects on peritoneal, medullary and J774 cells. G8 macrophages.

The use of medicinal plants as raw material for extracts production and pure substances isolation and subsequence development of new drugs represents a constantly growing area. However, some stages are indispensable before pharmacologically evaluating natural products such as medicines. Toxicity tests in mammalian cells are essential to initiate new drugs development or verify the substance's biocompatibility. Thus, we verified the toxicity of crude extracts and fractions with different polarities obtained from the leaves and stems of eight plant species. The toxic effect was evaluated on macrophages obtained from the bone marrow and peritoneal cavity of a Swiss webster mouse and J774 macrophages. G8 cell lineage. These macrophages were cultured in a 96-well plate, and the compounds were added at a concentration of 100 µg/mL for 24 hours. After this time, the supernatant was removed. The toxicity was evaluated for lactate dehydrogenase (LDH) release assay and the resazurin assay, which uses an indicator dye to measure oxidation-reduction reactions. The results showed a difference in the percentage of toxicity when comparing the same extract in different types of macrophages. This outcome indicates that these cells from different origins may exhibit different responses when exposed to the same natural compounds.

Sensitivity and specificity of serological tests, histopathology and immunohistochemistry for detection of Toxoplasma gondii infection in domestic chickens.

Since free-range chickens are important for the epidemiology of toxoplasmosis, this study evaluated the sensitivity and specificity of different laboratory techniques for the diagnosis of Toxoplasma gondii in these animals. Serum samples from 135 adult domestic chickens were tested for anti-T. gondii antibodies by the indirect fluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), modified agglutination test (MAT), and indirect hemagglutination test (IHAT). Tissue samples from all animals were analyzed by histopathology, immunohistochemistry and mouse bioassay (gold standard). Fifty-four chickens were positive for T. gondii in the bioassay. The sensitivity and specificity of the different tests were, respectively, 85% and 56% for ELISA; 80% and 52% for IFAT; 76% and 68% for MAT; 61% and 80% for IHAT; 7% and 98% for immunohistochemistry, and 6% and 98% for histopathology. The MAT was the most effective method for the diagnosis of T. gondii infection in chickens, followed by ELISA. Histopathology and immunohistochemistry are useful tools for the diagnosis of T. gondii infection in chickens due to their specificity.

Acute acquired toxoplasmosis: clinical-laboratorial aspects and ophthalmologic evaluation in a cohort of immunocompetent patients.

Most cases of acute acquired toxoplasmosis (AAT) are oligosymptomatic and self-limited. Therefore, these infections rarely indicate treatment. Prospective studies of AAT patients are rare in the medical literature. The frequency of systemic manifestations has not been sufficiently studied. In order to search for risks factors for systemic and ocular involvement, 37 patients were submitted to a diagnostic investigative protocol. The most frequent findings were lymph node enlargement (94.6%), asthenia (86.5%), headache (70.3%), fever (67.6%) and weight loss (62.2%). Hepatomegaly and/or splenomegaly were present in 21.6% of cases (8/37). Liver transaminases were elevated in 11 patients (29.7%) and lactic dehydrogenase in 17 patients (45.9%). Anaemia was found in four patients (10.8%), leucopoenia in six patients (16.2%), lymphocytosis in 14 patients (37.8%) and thrombocytopenia in one patient (2.7%). Fundoscopic examination revealed retinochoroiditis in four patients (10.8%). No statistical association was found between any one morbidity and retinochoroiditis. Nevertheless, a significant association was found between the presence of more than eight morbidity features at evaluation and long-lasting disease. An ideal diagnostic protocol for AAT would include evidence of systemic involvement. Such a protocol could be used when planning treatment.

[Standardization of enzyme-linked immunosorbent assay ELISA to detect anti-Toxoplasma gondii IgM and IgG antibodies, and comparison with the indirect immunofluorescence technique]. / Padronização de ensaio imunoenzimático para pesquisa de anticorpos das classes IgM e IgG anti-Toxoplasma gondii e comparação com a técnica de imunofluorescência indireta.

Serology has been the most popular method to diagnose toxoplasmosis. Accordingly, this study standardizes an enzyme-linked immunosorbent assay (ELISA) and compares its results with the IFI technique. In the IgG detection test, the standardized technique presented a sensibility (S) of 96.77%, a specificity (SP) of 75%, with a positive predictive value (PPV) of 83.33%, a negative predictive value (NPV) of 94.74%, and an adjusted concordance (K) of 73.50%. The IFI exhibited 83.87% for S, 79.16% for SP, 83.81% for PPV, 79. 16% for NPV, and 63% for K. The rough concordance between these two tests (ELISA/IFI) was 88.35% for the IgG detection test and 81.55% for the IgM detection test. K was 70.82% and 1.31% for IgG and IgM, respectively, the correlation index (r) being 0.556 for IgG and -0. 023 for IgM. We can conclude that standardized ELISA-IgG is indicated in serologic selection processes, whereas the ELISA-IgM is not recommended for presenting low values for the adjusted concordance with the reference technique, which suggests not very reliable results.

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 (Rop2196-561). Humoral response during Toxoplasma infection of humans was analyzed by immunoglobulin G (IgG), IgA, and IgM enzyme-linked immunosorbent assay with Rop2196-561 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 Rop2196-561 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 Rop2196-561 would be suitable for use in diagnostic systems, in combination with other T. gondii antigens, to detect specific IgG, IgA, and IgM antibodies.

Screening for active toxoplasmosis in patients by DNA hybridization with the ABGTg7 probe in blood samples.

We report the potential use of a specific Toxoplasma gondii DNA probe (ABGTg7). We applied a dot blot hybridization assay to blood samples for the diagnosis of cerebral toxoplasmosis (CT), acute toxoplasmic lymphadenopathy (ATL), and disseminated toxoplasmosis in transplant recipients (TRs). We studied a total of 84 individuals: 38 patients and 46 controls. We found positive hybridization signals for 12 (66.7%) of 18 patients with confirmed CT, 9 (52.9%) of 17 patients with ATL, and 2 (66.7%) of 3 TRs. PCR assays were performed in parallel for patients with ATL, resulting in T. gondii DNA detection for 10 patients (58.8%). A comparative study between dot blot and PCR assays performed with the blood of mice that had been experimentally infected with tachyzoites gave similar results: 60 and 70% positive results, respectively. Finally, the sum of positive values obtained by both DNA tests (dot blot assay plus PCR) increased the rate of positivity for ATL patients to 76.4%. These results demonstrate that the T. gondii ABGTg7 repetitive DNA element is an additional useful resource for diagnosing Toxoplasma parasitemia in patients with CT and ATL and in TRs. Thus, our ABGTg7-based dot blot test may lead to an improvement in T. gondii detection methods in patients with acute toxoplasmosis.

Concomitant cases of acquired toxoplasmosis in children of a single family: evidence of reinfection.

During 1979, three brothers had antibody titers for toxoplasmosis of 1:1,024, 1:64, and 1:16, respectively, by IgG indirect immunofluorescent antibody (IgG-IFA) test. The first child also had a fever and lymphadenopathy. In August 1980 the three children had lymphadenopathy and IgG-IFA test titers between 1:4,096 and 1:16,000. Two other brothers, first examined at that time, had IgG-IFA test titers between 1:1,024 and 1:4,096, one with ascending titers and the other with IgM antibodies to Toxoplasma gondii. The latter had lymphadenopathy, fever, and hepatosplenomegaly. Clinical and serologic examinations during March, April, and September 1981 revealed good health and decreasing IgG-IFA test titers in most of the brothers. The simultaneous increase of antibody titers during August 1980 in the three initial patients lead to the consideration of a probable reinfection. A simultaneous reactivation of the disease was considered less probable because acute toxoplasmosis occurred in two other brothers at the same time.