Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.

Standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats

The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20% when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.

Differential effects of isoproterenol on the activity of angiotensin-converting enzyme in the rat heart and aorta

The excessive stimulation of beta-adrenergic receptors in the heart induces myocardial hypertrophy. There are several experimental data suggesting that this hypertrophy may also depend, at least partially, on the increase of local production of angiotensin II secondary to the activation of the cardiac renin-angiotensin system. In this study we investigated the effects of isoproterenol on the activity of angiotensin-converting enzyme (ACE) in the heart and also in the aorta and plasma. Male Wistar rats weighing 250 to 305 g were treated with a dose of (±)-isoproterenol (0.3 mg kg-1 day-1, N = 8) sufficient to produce cardiac hypertrophy without deleterious effects on the pumping capacity of the heart. Control rats (N = 7) were treated with vehicle (corn oil). The animals were killed one week later. ACE activity was determined in vitro in the four cardiac chambers, aorta and plasma by a fluorimetric assay. A significant hypertrophy was observed in both ventricular chambers. ACE activity in the atria remained constant after isoproterenol treatment. There was a significant increase (P<0.05) of ACE activity in the right ventricle (6.9 ± 0.9 to 8.2 ± 0.6 nmol His-Leu g-1 min-1) and in the left ventricle (6.4 ± 1.1 to 8.9 ± 0.8 nmol His-Leu g-1 min-1). In the aorta, however, ACE activity decreased (P<0.01) after isoproterenol (41 ± 3 to 27 ± 2 nmol His-Leu g-1 min-1) while it remained unchanged in the plasma. These data suggest that ACE expression in the heart can be increased by stimulation of beta-adrenoceptors. However, this effect is not observed on other local renin-angiotensin systems, such as the aorta. Our data also suggest that the increased sympathetic discharge and the elevated plasma concentration of catecholamines may contribute to the upregulation of ACE expression in the heart after myocardial infarction and heart failure.

Increased levels of glutamate in the central nervous system are associated with behavioral symptoms in experimental malaria

Cerebral malaria (CM) is a severe complication resulting from Plasmodium falciparum infection. This condition has been associated with cognitive, behavioral and motor dysfunctions, seizures and coma. The underlying mechanisms of CM are incompletely understood. Glutamate and other metabolites such as lactate have been implicated in its pathogenesis. In the present study, we investigated the involvement of glutamate in the behavioral symptoms of CM. Seventeen female C57BL/6 mice (20-25 g) aged 6-8 weeks were infected with P. berghei ANKA by the intraperitoneal route using a standardized inoculation of 10(6) parasitized red blood cells suspended in 0.2 mL PBS. Control animals (N = 17) received the same volume of PBS. Behavioral and neurological symptoms were analyzed by the SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment (SHIRPA) battery. Glutamate release was measured in the cerebral cortex and cerebrospinal fluid of infected and control mice by fluorimetric assay. All functional categories of the SHIRPA battery were significantly altered in the infected mice at 6 days post-infection (dpi) (P ≤ 0.05). In parallel to CM symptoms, we found a significant increase in glutamate levels in the cerebral cortex (mean ± SEM; control: 11.62 ± 0.90 nmol/mg protein; infected at 3 dpi: 10.36 ± 1.17 nmol/mg protein; infected at 6 dpi: 26.65 ± 0.73 nmol/mg protein; with EGTA, control: 5.60 ± 1.92 nmol/mg protein; infected at 3 dpi: 6.24 ± 1.87 nmol/mg protein; infected at 6 dpi: 14.14 ± 0.84 nmol/mg protein) and in the cerebrospinal fluid (control: 128 ± 51.23 pmol/mg protein; infected: 301.4 ± 22.52 pmol/mg protein) of infected mice (P ≤ 0.05). These findings suggest a role of glutamate in the central nervous system dysfunction found in CM.

Anti-leishmania igA immunoenzymatic assay in mucocutaneous leishmaniasis (Preliminary report)

The Authors describe an anti-Leishmania IgA-ELISA assay in mucocutaneous leishmaniasis. Increased titers were found in leishmaniasis patients, mainly in the first and second year of infection and in deep mycoses patients showing either mucosal involvement or widespread disease.

An immunohistochemic assay to localize leptospires in tissue specimens

An Immunoperoxidase technique for identification of leptospires in formalin fixed, paraffin embedded kidney sections is presented, using peroxidase-antiperoxidase complex. The anti-leptospiral antibody was raised in rabbit. Possible applications of this technique are discussed.

Enzyme linked immunosorbent assay: determination of anti-adenovirus antibodies in an infant population

In order to define an accurate assay for anti-adenovirus antibody detection, a recently developed ELISA was compared with IFA and CF. On 58 sera, the ELISA was more sensitive than both CF and IFA, which showed relative sensitivities of 63% and 94%, respectively. It was not possible to determine the exact specificity of the tests because of the lack of a gold standard. Furthermore, the ELISA was used to define the prevalence of adenovirus antibodies in 116 infants between 1 and 24 months old (mean 7.28). The data showed that maternal antibodies waned by the age of 5 to 6 months and that more than 80% of the children had been infected by adenoviruses by the age of 10 months.

Screening the mutagenic activities of coomonly used antiparasite drugs by the Simultest, a simplified Salmonella/microsome plate incorporation assay

The mutagenic activities of 16 anti-parasite drugs were screened by the Simultest in both qualitative (spot test) and quantitative (plate incorporation) assays with a Salmonella typhimurium pool composed by the indicator strains TA97, TA 98, TA100 and TA102. Four anti Chagas' disease drugs (nifurtimox, benznidazole, CL 64,855, and MK 436) and two anti-amebae drugs (metronidazole and tinidazole) gave positive results in qualitative tests and incorporation of rat liver microsomes did not alter the results. Comparative dose response curves of the mutagenic activities of CL 64,855, metronidazole and benznidazole obtained by the simultest and by individual Salmonella indicator strains demonstrated that both approachs have similar sensitivities. The results corroborate the validity of the Simultest, as a simplified, fast and economic version of the Ames test in preliminary screening of potential mutagenic drugs.

Enzyme-linked immunosorbent assay (ELISA) for measles antibody: a comparison with haemagglutination inhibition, immunofluorescence and plaque neutralization tests

An enzyme-linked immunosorbent assay (ELISA) for measles antibodies was compared with Plaque Neutralization (PRN), Haemagglutination inhibition (HI) and Fluorescent antibody (IFA) tests in 181 sera from vaccinated children and umbilical cord. Of 179 positive samples by the sensitive PRN, only two, with titers of 8, were negative by ELISA (copositivity of 98.9%). IFA and HI presented, respectively, copo-sitivities of 93.3% and 82.7%. The ELISA presented a high sensitivity as well as a good reproducibility and represents an alternative for the time consuming PRN for detection of low measles antibodies.

Performance indexes of a dot-enzime-linked immunosorbent assay (dot-ELISA) and an ezyme-linked immunosorbent assay (IgG-ELISA) for field surveys of new world leishmaniasis

Diagnostic performance indexes of sensitivity, specificity, positive predictive value and efficiency were determined for dot-ELISA and IgG-ELISA tests in 340 leishmaniasis sera. Sensitivity of the dot-ELISA was significantly lower than IgG-ELISA's; the two tests had indexes of specificity and positive predictive value of the same magnitude. Seventy-eight sera gave a negative dot-ELISA test result and a positive IgG-ELISA test result. When sera were classified according to different criteria as how to interpret this diversity, the kappa statistic did not corroborate the classification indicating that the two tests display a substantial strength of agreement. The results presented indicate that performance indexes accrued in a survey where variables arc well known may be extrapolated to other population studies if the disease presents itself as highly prevalent (due to a selection bias or not) and may be expected to discriminate a disease status among test positives.

Evolution of subpatent parasitaemia in Trypanosoma cruzi chronically infected mice with the help of a cyclophosphamide amplification transfer assay

We have evaluated the sensitivity of the classical blood subinoculation method, modified through cyclophosphamide treatment of transferred mice, for the detection of occult parasitaemias in Trypanosoma cruzi chronically infected mice. Besides its simplicity, the method was shown to be highly sensitive for both the "chronic" phase parasites (99% of chronic cases were shown to harbour occult parasitaemias) and for the acute phase parasites (T. cruzi could be detected in 53.8% of animals transferred with one Y strain parasite and in 20% of animals transferred with one CL strain parasite). Using acute phase bloodforms, the assay proved to be more sensitive than conventional subinoculation when dealing with the CL, but not the Y strain of the parasite. With the help of this parasite detection tool, we have studied during a one year period, the evolution of subpatent parasitaemias in a group of mice which survived through chemotherapy from lethal acute phase of T. cruzi infection. Cyclophosphamide transfer assay revealed occult parasitaemias in 100% of the chronic animals, nevertheless, continuous and discontinuous patterns of positivity were observed.

Standardization of dot-ELISA for the serological diagnosis of toxocariasis and comparision of the assay with ELISA

The dot-enzyme-linked immunosorbent assay (dot-ELISA) was standardized using somatic (S) and excretory-secretory (ES) antigens of Toxocara-canis for the detection of specific antibodies in 22 serum samples from children aged 1 to 15 years, with clinical signs of toxocariasis. Fourteen serum samples from apparently normal individuals and 28 sera from patients with other pathologies were used as controls. All samples were used before and after absorption with Ascaris suum extract. When the results were evaluated in comparison with ELISA, the two tests were found to have similar sensitivity, but dot-ELISA was found to be more specific in the presence of the two antigens studied. Dot-ELISA proved to be effective for the diagnosis of human toxocariasis, presenting advantages in terms of yield, stability, time and ease of execution and low cost.

Enzyme-linked immunosorbent assay ELISA for the detection of antibodies in the human leptospirosis

An Enzyme-linked immunosorbent assay ELISA was evaluated for the detection of IgA antibodies in the human leptospirosis. The assay proved to be sensitive and specific when compared with the ELISA-IgM, in the examinated serum samples. The results found suggest that IgA antibodies became positive later in leptospirosis, and will can be an evolutive indicator in the development of the disease

Gelatin particle indirect agglutination test for serodiagnosis of schistosomiasis: comparative study with enzyme-linked immunosorbent assay

A new serological test, the gelatin particle agglutination test (GPAT), was used for the serodiagnosis of schistosomiasis mansoni. This technique showed the sensitivity (90.6%) and specificity (97.8%) close to those of enzyme-linked immunosorbent assay. The GPAT can be easily and rapidly performed without specialized equipment, by using lyophilized antigen-coated gelatin particles. The test also seems to be useful for mass screening of Schistosoma infection in field conditions.

Dot-enzyme-linked immunosorbent assay (Dot-ELISA) for detection of pneumococcal polysaccharide antigens in pleural fluid effusion samples.: Comparison with bacterial culture, counterimmunoelectrophoresis and latex agglutination

A dot-enzyme-linked immunosorbent assay (Dot-ELISA) for pneumococcal antigen detection was standardized in view of the need for a rapid and accurate immunodiagnosis of acute pneumococcal pneumonia. A total of 442 pleural fluid effusion samples (PFES) from children with clinical and laboratory diagnoses of acute bacterial pneumonia, plus 38 control PFES from tuberculosis patients and 20 negative control serum samples from healthy children were evaluated by Dot-ELISA. The samples were previously treated with 0.1 M EDTA pH 7.5 at 90°C for 10 min and dotted on nitrocellulose membrane. Pneumococcal omniserum diluted at 1:200 was employed in this assay for antigen detection. When compared with standard bacterial culture, counterimmunoelectrophoresis and latex agglutination techniques, the Dot-ELISA results showed relative indices of 0.940 to sensitivity, 0.830 to specificity and 0.760 to agreement. Pneumococcal omniserum proved to be an optimal polyvalent antiserum for the detection of pneumococcal antigen by Dot-ELISA. Dot-ELISA proved to be a practical alternative technique for the diagnosis of pneumococcal pneumonia.