Measurement of antigen specific lymphocyte proliferation using 5-bromo-deoxyuridine incorporation. An easy and low cost alternative to radioactive thymidine incorporation.

The classical in vitro assay for the determination of cell mediated immune responses is the lymphocyte transformation test (LTT) in which cell proliferation is measured by incorporation of radioactive labeled thymidine (3H-TdR). The LTT assay using 3H-TdR is less suited for modestly equipped laboratories as it is costly, laborious and involves the need to handle radioactive isotopes and specialized equipment. Here we describe an improved alternative LTT method which is capable of detecting specific cellular immune reactions (CMI) against (mycobacterial) antigens in vitro. This assay, the bromodeoxyuridine-ELISA LTT test, is simple, less expensive, reproducible and is as sensitive as the 3H-TdR test. The specific advantages of the test are a simple denaturation step and the fact that no radioactive isotopes are needed. The test is specifically suited for research laboratories in tropical countries which study CMI in those human infectious diseases where this arm of the immune response plays a pivotal role in the generation of immunity, e.g., in tuberculosis, leprosy and leishmaniasis.

Dissecting the IgM antibody response during the acute and latent phase of toxoplasmosis.

A major problem in anti-toxoplasma IgM serology is the occurrence of clinically non-relevant (CNR) IgM in sera of latently infected (LI) individuals. The susceptibility for CNR IgM of the Toxo ISAGA IgM, Platelia Toxo IgM and Vidas Toxo IgM assays was determined using sera of LI individuals with an IgM titer in the Abbott IMx Toxo IgM assay. The specificity of CNR-IgM and IgM antibodies in sera of acutely infected (AI) individuals was compared by immunoblotting.Seven/19 samples with CNR IgM antibodies were found positive in the ISAGA IgM, compared with 16/19 and 17/19 with the Vidas and Platelia Toxo IgM assays, respectively. In contrast, immunoblotting allowed a distinction between CNR IgM and AI IgM antibodies of the 30 sera studied.Clearly, IgM assays are susceptible to CNR IgM antibodies. In cases of doubt, immunoblotting is of value as confirmation method. Because CNR IgM antibodies are specific for toxoplasma, it will be difficult to improve IgM ELISAs in such a way that CNR IgM antibodies will not interfere with the analysis.

Evaluation of the Eiken latex agglutination test for anti-Toxoplasma antibodies and seroprevalence of Toxoplasma infection among factory workers in Addis Ababa, Ethiopia.

Sera from 170 factory workers aged 18-45 years enrolled in a pilot study of human immunodeficiency virus 1 (HIV-1) infection in Addis Ababa, Ethiopia, were screened for anti-Toxoplasma immunoglobulin G antibodies by the Sabin-Feldman test (reference standard) and the Eiken latex agglutination test (under evaluation for use in developing countries). Based on the Sabin-Feldman test, the prevalence of anti-Toxoplasma antibodies was 80.0% (95% confidence interval 73.9-86.1%). The sensitivity and specificity of the Eiken latex agglutination test were 96.3% and 97.1%, respectively, showing its validity for the detection of anti-Toxoplasma antibodies. The prevalence of antibodies did not differ between individuals infected and uninfected with HIV-1 (74.2% versus 83.3%, P > 0.05). However, antibody titres were higher in HIV-infected persons than in those who were uninfected (P < 0.001). Based on these findings, we expect that toxoplasmic encephalitis will be a common opportunistic infection among HIV-infected Ethiopians, and chemoprophylaxis with co-trimoxazole may be beneficial to those with low CD4+ T cell counts. The prognostic significance of high titres of anti-Toxoplasma antibodies remains to be established among Ethiopian HIV-infected individuals.

Production and characterization of monoclonal antibodies against specific serotypes of Mycobacterium avium and the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex.

Serotype-specific and Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex (MAIS complex)-specific monoclonal antibodies (MAbs) were prepared. A series of MAbs were obtained, five specific for serotype 2, three specific for serotype 4, eight against a strain of serotype 19, two specific for the MAIS complex, and two against a common glycolipid shared by all the mycobacteria tested so far. The serotype-specific and the MAIS complex-specific MAbs reacted in immunofluorescence with intact mycobacteria and in enzyme-linked immunosorbent assay and immuno-thin-layer chromatography with lipid extracts of mycobacteria. The two MAbs against a common mycobacterial glycolipid reacted only in lipid enzyme-linked immunosorbent assay and immuno-thin-layer chromatography. All MAbs were directed against glycopeptidolipids (GPLs), except for four MAbs against proteins of serotype 19. The serotype- and MAIS complex-specific epitopes on GPLs are exposed on the mycobacterial cell wall, in contrast with the common mycobacterial glycolipid, which is probably located inside the cell wall. The serotype-specific MAbs reacted with native as well as deacetylated GPLs, in contrast with the MAIS complex-specific MAbs, which reacted only with native GPLs. The MAbs will be useful for the identification of MAIS complex and M. avium serotypes 2 and 4 and a strain of serotype 19, GPL analyses with immuno-thin-layer chromatography, and the localization of GPL epitopes in mycobacteria.

Septata intestinalis frequently isolated from stool of AIDS patients with a new cultivation method.

Two species of microsporidia, Enterocytozoon bieneusi and Septata intestinalis have been reported as intestinal parasites of AIDS patients. In attempts to establish E. bieneusi in vitro, spores were concentrated from stool samples from 4 AIDS patients with biopsy-proven E. bieneusi infections. After sterilization of the concentrate in antibiotic solution, the spores were added to monolayers of RK13 cells grown on the membranes of Transwells. Cultures were established from 7 stool samples from the 4 patients but in every case the species established was S. intestinalis not E. bieneusi. On retrospective examination of the stools, a very small number of spores of a size comparable to that of S. intestinalis was found but this species was not detected in biopsies. Typical septate vacuoles containing Type I tubules were observed in vitro but in contrast to the original description, meronts were intravacuolar and sporogony was mainly disporoblastic. The cultivation system, used for the first time for microsporidia, revealed the presence of unsuspected S. intestinalis infections and indicates that this species may be much more common than hitherto suspected. S. intestinalis has not previously been cultured.