CL 64, 855, a potent do anti-Trypanosoma cruzi drug, is also mutagenic in the salmonella/microsome assay

The nitroimidazole-tiadiazole derivative CL 64,855 (2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole, a potent anti-trypanosomal drug, was assayed in a short-term bacterial mutagenicity test with Salmonella typhimurium strains TA 98, TA 100 and TA 102. Results indicate that CL 64,855 is a potent frameshift mutagen detected by strains TA 98 and TA 102. CL 64,855 was able to revert the indicators strains at concentrations as low as 0.1 µg/plate. Metabolic activation experiments with rat liver microsomal fractions did not increase the mutagenic action of Cl 64,855.

Effect of drugs on Trypanosoma cruzi and on its interaction with heart muscle cell in vitro

Megazol, nifurtimox, benznidazol and allopurinol were investigated, by light and electron µscopy, for their action on T. cruzi. Both the direct effect upon amastigote and trypomastigote forms and the effect upon the interaction of heart muscle cells (HMC) with bloodstream trypomastigotes were studied. The proliferation of amastigotes in Warren medium was inhibited in a dose-dependent manner by megazol, nifurtimox and benznidazol. Treatment of amastigotes (25-50 µM/24 h) and trypomastigotes (25 µM/24h) led to several ultrastructural alterations in the parasites. These three drugs also had a potent effect on the treatment of infected heart muscle cells when added at the beginning of the interaction or after one or three days of infection. The interiorized parasites showed a similar pattern of ultrastructural alterations as observed by the direct effect on the amastigotes. The primary heart muscle cell culture proved to be a suitable model for the study of drugs on intracellular parasites. Likewise, the amastigote proliferation in axenic medium was shown to be an adequate assay for an initial trial of drugs. These parameters seem very reliable to us for a systematic investigation of the mechanism of action of new drugs.

The use of dacron plates for DOT enzyme-linked immunosorbent assay (DOT-ELISA)

Dacron (polyethylenetherephthalate) is proposed as a matrix for dot-ELISA procedures, as an alternative to nitrocellulose. Plates of dacron were partially hydrazinolyzed and hydrazide groups introduced were converted to azide groups. The derivative dacron-antigen was covalently linked on to the plates through these azide groups. The derivative dacron-antigen was exaustively washed according to CROOK and antigen was still fixed onto the plates. Protein F1A purified from Yersinia pestis was used as a model. Triration of sera from immunized and non immunized rabbits against this protein was carried out by employing the dot-ELISA method. No significant difference was observed using dacron-antigen and nitrocellulose-antigen preparations. However, both procedures showed to have a significant better performance in comparasion with the passive hemagglutination method. The specificity and reproductibility of the dot-ELISA assay using both preparations showed a similar behaviour. Nitrocellulose preparation was stable at 4ºC, 28ºC and -20ºC for 90 days, whereas the dacron-antigen derivative was stable only when stored at 4ºC. Dacron-antigen derivative could be re-used when the spot developing was proceeded using 4-chloro-1-naphtol as substrate.

Development of an immunoenzymatic assay using a monoclonal antibody against a 50-kDa catabolite from the P126 Plasmodium falciparum protein to the diagnosis of malaria infection

The WHO criterion of defering any donation of blood by a confirmed case of malaria for three years after cessation of therapy can not be applied in areas where malaria in endemic. For this reason we developed an immunoenzymatic assay for the detection of plasmodial antigens for blood screening in malararial endemic areas. So, we tested sera from 191 individuals. Among patients with active disease 100% of the cases of Plasmodium falciparum or mixed infections and 91.7% of those with P. vivax were positive for the presence of plasmodial antigens. The lower parasitaemia detected was 0.0003% for P. vivax malária. When the frequency of positive circulating malarial antigens was evaluated among asymptomatic and symptomatic individuals with negative TBS, positive results were found in respectively 38.7% and 17.7% of the individuals studied in the 30 days after confirmed malaria attack. Data provide by these assays have shown that ELISA seemed to be more sensitive than parasitological examination for malaria diagnosis. This test by virtue of its high sensivity and the facilities in processing a large number of specimens, can prove to be useful in endemic areas for the recognition of asymptomatic malaria and screening of blood donors.

Standardization of the dot enzyme-lynked immunosorbent assay (dot-ELISA) for experimental plague

A dot enzyme linked immunosorbent assay (dot-ELISA) was previously developed to detect specific antibodies in rabbits sera immunized against FIA protein obtained from Yersina pestis. This antigen was covalently linked onto the surface of dacron (polyethyleneterephthalate). Here, standard conditions are described for the optimization of this procedure: an amount of 20 ng of FIA protein was fixed onto dacron; anti-rabbit IgG peroxidase conjugate diluted 1:8,000 and 30% non-fat instant milk as blocking substance were used throughout the method. This procedure was compared with that employing nitrocellulose as solid-phase which showed to be more sensitive. However, the method based on dacron did not show false positive reactions against non-immunized rabbits sera at low antigen amount and diluted anti-IgG peroxidase conjugate.

An experimental and clinical assay with ketoconazole in the treatment of Chagas disease

Ketoconazole an azole antifungic drug which is already in the market has also been demonstrated to be active against Trypanossoma cruzi experimental infections. In this paper we confirmed the drug effect and investigated its range of activity against different T. cruzi strains naturally resistant or susceptible to both standard drugs Nifurtimox and Benznidazole used clinically in Chagas disease. Moreover, we have shown that the association of Ketoconazole plus Lovastatin (an inhibitor of sterol synthesis), which has an antiproliferative effect against T. cruzi in vitro, failed to enhance the supressive effect of Ketoconazole displayed when administered alone to infected mice. Finally, administration in chronic chagasic patients of Ketoconazole at doses used in the treatment of deep mycosis also failed to induce cure as demonstrated by parasitological and serological tests. The strategy of identify and test drugs which are already in the market and fortuitously are active against T. cruzi has been discussed.

Improved enzyme-linked immunoadsorbent assay (ELISA) for the study of Trypanosoma cruzi-host cell interaction in vitro

We herein present an improved assay for detecting the presence of Trypanosoma cruzi in infected cultures. Using chagasic human sera (CHS), we were able to detect T. cruzi infection in primary cultures of both peritoneal macrophages and heart muscle cells (MHC). To avoid elevated background levels - hitherto observed in all experiments especially in those using HMC - CHS were preincubated with uninfected cells in monolayers or suspensions prior to being used for detection of T. cruzi in infected monolayers. Preincubation with cell suspensions gave better results than with monolayers, reducing background by up to three times and increasing sensitivity by to twenty times. In addition, the continous fibroplastic cell line L929 was shown to be suitable for preadsorption of CHS. These results indicate that the high background levels observed in previous reports may be due to the presence of human autoantibodies that recognize surface and/or extracellular matrix components in cell monolayers. We therefore propose a modified procedure that increases the performance of the ELISA method, making it an useful tool even in cultures that would otherwise be expected to present low levels of infection or high levels of background

Comparative evaluation of a simple and sensitive assay for detection of orthomyxo and paramyxoviruses

Studies were done to evaluate comparatively the traditional HA assay and a more recently introduced lectin-neuraminidase (LN) methodologyin search of a simple and sensitive assay for virus detection during laboratorial diagnosis. The results proved the value of LN assay as a sensitive methodologyfor detection of virus particles, presenting results at least equal to those obtained by HA (hemagglutination) assay, with significant values of accumulated frequencies for LN/HA factors (ratios between LN and HA titers) higher than two. The accumulated values of frequencies for LN/HA factors as high as four were very significant, 72.7 (per cent) for influenzavirus and 60.7 (per cent) for Newcastle disease virus (NDV), moreover accumulated frequencies for LN/HA factors even as high as 32 were due to influenzavirus (45.4 per cent) and NDV (7.2 per cent) samples. After the storage period, most of those concentraded samples that even did not present HA titers could be detected through LN assay, demonstrating a lower threshold for virus detection.

Immunogenicity of multiple antigen peptides containing Plasmodium vivax CS epitopes in BALB/c mice

Multiple antigen peptide systems (MAPs) allow the incorporation of various epitopes in to a single synthetic peptide immunogen. We have characterized the immune response of BALB/c mice to a series of MAPs assembled with different B and T cell epitopes derived from the Plasmodium vivax circumsporozoite (CS) protein. A B-cell epitope from the central repeat domain and two T-cell epitopes from the amino and carboxyl flanking regions were used to assembled eight different MAPs. An additional universal T cell epitope (ptt-30) from tetanus toxin protein was included. Immunogenicity in terms of antibody responses and in vitro T lymphocyte proliferation was evaluated. MAPs containing B and T cell epitopes induced high titers of anti-peptides antibodies, which recognized the native protein on sporozoites as determined by IFAT. The antibody specificity was also determined by a competitive inhibition assay with different MAPs. A MAP containing the B cell epitope (p11) and the universal epitope ptt-30 together with another composed of p11 and the promiscuous T cell epitope (p25) proved to be the most immunogenic. The strong antibody response and specificity for the cognate protein indicates that further studies designed to assess the potential of these proteins as human malaria vaccine candidates are warranted.