Seroprevalence to Trypanosoma cruzi in rural communities of the state of Querétaro (Mexico): statistical evaluation of tests.

OBJECTIVES: The detection of anti-Trypanosoma cruzi antibodies has become one of the priorities of the clinical establishments in the health sector, due both to the increase in positive cases found in transfusion centres as well as to the appearance of patients with characteristic Chagas cardiopathies that seek emergency treatment in the main hospitals of Querétaro (Mexico). DESIGN AND METHODS: The present study seeks to establish for the first time the infection level of Trypanosoma cruzi, in the rural communities of this state and implement the preventive measures necessary to control and/or eradicate this infection. A transversal study was conducted, examining seriologically 1029 blood samples of the inhabitants of rural areas of the state of Querétaro, to detect anti-Trypanosoma cruzi antibodies. RESULTS: The indirect serological diagnostic tests were indirect hemagglutination, enzymo-immunoenzymatic absorbent, recombinant ELISA, and indirect immunofluorescence. For the diagnostic evaluation of serological tests used, ELISA was considered the control test. CONCLUSIONS: The first conclusion was that the two tests with the greatest serological reactivity were ELISA and recombinant ELISA, followed by IFA and IHA, respectively, with the final percentage of positives being 6.6%, far above the national mean of seroprevalence in Mexico (1.6%). On the other hand, the sensitivity, specificity, VP+, VP-, percentage of concordance and Kappa index of the recombinant ELISA tests, IFA, and IHA were determined against the control ELISA. It was found that ELISA and recombinant ELISA presented a greater sensitivity level, as well as the highest values for the different parameters studied.

The use of an excreted superoxide dismutase in an ELISA and Western blotting for the diagnosis of Leishmania (Leishmania) infantum naturally infected dogs.

An excreted iron superoxide dismutase of pI 3.75 and a molecular mass of approximately 25 kDa was partially purified by QAE Sephadex ion-exchange chromatography from the in vitro culture of Leishmania (Leishmania) infantum. This enzyme was detected by enzyme-linked immunosorbent assay and Western blot of anti-L. infantum antibodies in dog serum. For the determination of the sensitivity and specificity of this protein, the results using the complete-parasite antigen fraction were taken as references. For this, 39 sera were assayed in dogs from different Spanish provinces. By Western blot, at a dilution of 1:250, 82% of the sera were positive when superoxide dismutase excreted was used as the antigen, against 56.4% positivity when the complete parasite was used as the antigen. These findings support the results of a previous study, indicating that the superoxide dismutase excreted can be useful in diagnosing L. (L.) infantum.

The prevalence of enteropathy due to strongyloidiasis in Puerto Maldonado (Peruvian Amazon).

Human strongyloidiasis is an important health problem in the southeast region of Peruvian Amazon, due to its prevalence and long term morbidity. An epidemiological study was conducted in the Peruvian Amazon area of Puerto Maldonado to determine the prevalence of strongyloidiasis in the population. Stool samples were collected from 1,133 patients at the outpatient department of our clinic. Strongyloidiasis affected 221 examined patients (20%). Prevalence was highest in males, mostly in children and elderly men. People living in urban and marginal urban areas, those coming from outside the region, and Andean people, showed the highest prevalences. Pre-school children were more likely to be parasitized than older children. The most common symptoms were diarrhea (55%), abdominal pain (32%) and cough (53%). One in 7 (13%) affected patients presented with moderate or severe symptoms, including life-threatening complications. Other intestinal parasites were found frequently in patients diagnosed with strongyloidiasis. Improved human waste disposal services are considered to be the main requirement to reduce the high prevalence of this disease.

The prevalence of enteropathy due to strongyloidiasis in Puerto Maldonado (Peruvian Amazon)

Human strongyloidiasis is an important health problem in the southeast region of Peruvian Amazon, due to its prevalence and long term morbidade. An epidemiological study was conducted in the Peruvian Amazon area of Puerto Maldonado to determine the prevalence of strongyloidiasis in the population. Stool samples were collected from 1,133 patients at the outpatient department of our clinic. Strongyloidiasis affected 221 examined patients (20 percent). Prevalence was highest in males, mostly in children and elderly men. People living in urban and marginal urban areas, those coming from outside the region, and Andean people, showed the highest prevalences. Pre-school children were more likely to be parasitized than older children. The most common symptoms were diarrhea (55 percent), abdominal pain (32 percent) and cough (53 percent). One in 7 (13 percent) affected patients presented with moderate or severe symptoms, including life-threatening complications. Other intestinal parasites were found frequently in patients diagnosed with strongyloidiasis. Improved human waste disposal services are considered to be main requirement to reduce the high prevalence of this disease.

Proton nuclear magnetic resonance analysis of metabolic end products of the Bolivia strain of Trypanosoma cruzi and three of its clones.

Proton nuclear magnetic resonance (1H NMR) was used to study the in vivo metabolism of Trypanosoma cruzi, the pathogen causing American trypanosomiasis (Chagas' disease). Three clones were isolated from a strain of T. cruzi (Bolivia strain), The clones I, II and III and the original strain were characterized according to the spectra of their metabolic pathways to test the hypothesis that clonal evolution of T. cruzi has a major impact on biologically relevant properties of this parasite. T. cruzi (Bolivia strain) excreted acetate, alanine, glycerol, and succinate as major end products, in the proportion 6:4:2:2. Comparing the spectra of T. cruzi clones with the original Bolivia strain revealed both quantitative, as well as qualitative differences in the metabolites excreted: the clones I and II, as opposed to the Bolivia strain and clone III, excreted significant quantities of ethanol.

Is the malaria diagnosis expensive?

Malaria remains the most important of the tropical diseases, widespread throughout the tropics, but also occurring in many temperate regions. The disease causes a heavy toll of illness and death, among children in endemic areas. It also poses a risk to business travellers, tourists and immigrants and imported cases of malaria are increasingly seen in non-endemic areas. We discuss here how microscopical diagnosis is essential for identifying Plasmodium species responsible of the infection and discarding possible mixed infections. Thus, a correct treatment can be administered in 30 min, avoiding secondary stays and saving important amounts of money. Problems of drug resistance have to be distinguished from those arising due to erroneous diagnosis.

Relationships between histopathological findings and phylogenetic divergence in Trypanosoma cruzi.

Problems have been raised by natural genetic diversity of Trypanosoma cruzi, the causal agent of Chagas' disease, and other protozoa in terms of both basic and applied science. T. cruzi manifests a great diversity of medical and biological properties which could be the origin of clinical variability in the disease. We propose possible correlations between genetic distances, or phylogenetic divergence, and histopathological data. To ascertain this aspect, 15 cloned stocks pertaining to three major clones or genotypes (19, 20 and 39) were compared. Sets of 24 mice infected with each stock were studied for histopathological lesions. Brain, heart, lung, liver, spleen, urinary bladder, bone marrow, colon, kidney and skeletal muscle were extracted from each mouse. Qualitative and quantitative differences showed at histopathological examination. An important encephalic softening was found in brains of most mice infected by genotype 20, corresponding to areas of inflammation and liquified necrosis. Other inflammatory tissue lesions in the histological sections of the three genotypes were similar. Skeletal muscle tropism was higher than cardiac tropism in all the studied genotypes. All three genotypes shared parasite presence in skeletal muscle. Differences related to cardiac tropism were important: in genotype 19, 50% of studied stocks presented pseudocysts; 20% in genotype 20 and 83% in genotype 39. Parasite presence in other tissues was scanty: in brain only in genotype 20 and in spleen and liver only in genotype 39. We found important histopathogenicity differences among the three studied genotypes, but they do not support the hypothesis of zymodeme pathogenic specificity due to the great diversity among stocks within each genotype.

Are genotypes of Trypanosoma cruzi involved in the challenge of chagasic cardiomyopathy?

Myocardial damage in Chagas' disease differs, depending on the particular Trypanosoma cruzi stock. It is reasonable to expect that the extent of phylogenetic divergence between lineages will have an impact on the biological properties of the parasite. The aim of the present work was to evaluate this impact on the cardiac damage produced by this protozoan. Heart histopathologic lesions were studied in mice infected with 15 cloned stocks of T. cruzi of various origins pertaining to 3 major clones or genotypes (19, 20, and 39) that share 3 different profiles for a given set of genetic markers. Sets of mice were infected intraperitoneally with 10(6) blood trypomastigotes of each of the T. cruzi clones. The macroscopy study showed a cardiac index (CI) higher than 0.6 (cardiomegaly) in 5 of the 15 stocks studied (33.33%). Inflammatory infiltrates appeared in stocks pertaining to the three genotypes studied without relation to the genetic pattern. Pseudocysts were present at higher levels (83%) in stocks pertaining to genotype 39. A lower rate could be seen in stocks pertaining to genotype 19 (50%). Only one stock pertaining to genotype 20 presented myocardial parasites (20%). Hearts were also studied for lesions in the different cardiac chambers. Inflammatory foci as well as pseudocysts appeared mainly in ventricles, with the left ventricle sharing the highest percentage of pathologic findings. In summary, in spite of the similar inflammatory pattern demonstrated for all stocks studied, the parasite's presence seemed to be related to the genotype of reference, but no relation could be demonstrated with the genetic distances.

Characterization of a Trypanosoma rangeli strain of Colombian origin.

A Colombian strain of Trypanosoma rangeli was characterized by analyzing its behaviour in different axenic and cellular culture, its infection rate and the histopathological lesions produced in experimental animals. Although slight inflammatory infiltrations were shown in different histopathological sections, no pseudocysts could be observed. Grace's insect medium is better than liver infusion tryptose or artificial triatomine urine supplemented with proline when studying T. rangeli metacyclogenesis, with a peak of 32% trypomastigotes. High infection rates were found in VERO and J774 cells. Because of its 100% infectivity rates and adequacy of parasitemia levels, C23 strain is a suitable model of T. rangeli biology study.

Protective effect of Trypanosoma rangeli against infections with a highly virulent strain of Trypanosoma cruzi.

We investigated the protective effect of Trypanosoma rangeli against infection with Trypanosoma cruzi in animal models of various ages and with different doses of inoculum. The age of the mice and the dose of parasites determined the course of the infection. When T. cruzi was inoculated into mice after challenge with T. rangeli, parasitaemia was more controlled, mortality decreased and histopathology showed lower inflammatory infiltration and pseudocysts. This study proposes a new murine model of the protective effect of recombinant proteins of T. rangeli for possible application in the vaccines field.

Trypanosoma rangeli: increase in virulence with inocula of different origins in the experimental infection in mice.

We compared two murine models of Trypanosoma rangeli infection. The same inoculum dose and age-matched hosts were used in both cases. One group was infected with trypomastigotes obtained from passages in mice and the other, with trypomastigotes obtained from cell culture after a passage in mice. We observed that trypomastigotes obtained from the in vitro cellular infection showed increased virulence in experimental animals, with a 70% rate of death being noted in experimental mice instead of the lack of mortality seen when in vivo-derived parasites were used. The greatest levels of parasitemia and tissual lesions in the presence of the parasite also occurred when in vitro-derived parasites were used.

Morphological and morphometric variability of three clones of Trypanosoma cruzi and the strain of origin (Bolivia).

Knowing the great diversity of medical and biological properties of Trypanosoma cruzi, the causal agent of Chagas' disease, we quantified the morphological parameters that typify the different forms of three clones of T. cruzi and their original strain, Bolivia, in comparison among themselves and with strain Bolivia, attempting to provide additional data concerning the clonal biological behaviour of this parasite. Blood forms morphology was quantified using a computerized image analysis (Videoplan/Kontron) and statistical analysis was determined using ANOVA-1 Test. Large number of quantitative differences among slender, broad, and stout forms were found. The comparison of clones I, II and III with their mother strain, leads to the emergence of significant differences in at least 12 parameters out of the 16 we studied. When clones were compared among themselves, the differences decreased. Variations of the percentages of the three kinds of clones were found along the acute infection. These data are the first step in correlating the morphological and pathogenic characteristics of the parasite.

Biological comparison between three clones of Trypanosoma cruzi and the strain of origin (Bolivia) with reference to clonal evolution studies.

After isolating three clones of Trypanosoma cruzi (Bolivia), we first characterized them according to parasitaemia, pleomorphism and virulence, and then histopathologically. The study's interest lies on the hypothesis that clonal evolution of T. cruzi has a major impact on biologically relevant properties of this parasite. Data obtained from the studies of parasitaemia, pleomorphism and virulence showed no differences between the groups studied. As a final point, the histopathological study shows us a muscular tissue tropism both in clones and in their mother strain (Bolivia). In this paper, we conclude that Bolivia strain and clones isolated from it, pertaining to the same major clone share similar biological properties.

A clinical-parasitological monotherapy cure in the treatment of experimental infection by a highly virulent strain of Toxoplasma gondii.

Toxoplasmic encephalitis in patients with the acquired immunodeficiency syndrome (AIDS) is treated classically with pyrimethamine plus sulfadiazine. Unfortunately, up to 40% of these patients are unable to complete the course of therapy because of adverse reactions to sulfonamides. This study considers the possible usefulness of monotherapies in the treatment of acute toxoplasmosis, producing parasitological cures 2-3 months after the date of infection. With this therapy, the main adverse effects are suppressed. Groups of mice infected with the RH strain of Toxoplasma gondii were treated with pyrimethamine alone, sulfadiazine alone, and pyrimethamine plus sulfadiazine for 7 d. Treatment with pyrimethamine plus sulfadiazine produced clinical cures in 100% of the infected mice 1 month after infection. Treatment with pyrimethamine gave a 60% survival rate (clinical cure) at 1 month postinfection. Finally, treatment with sulfadiazine produced a 60% survival rate at 1 month postinfection. Although the antitoxoplasmic regimen with pyrimethamine plus sulfadiazine has proven to be effective in intensive treatment of toxoplasmic encephalitis, relapses occur in more than 80% of cases after cessation of antitoxoplasmic therapy, making secondary prophylaxis mandatory. In this study the efficacy of treatment was also evaluated in terms of parasitological cure. None of the three therapies showed parasitological cure after 1 month of treatment. When the intervals were extended to a 3-month observation, monotherapy with pyrimethamine and sulfadiazine alone produced a parasitological cure.

A new murine model of severe acute toxoplasma encephalitis.

We have developed a new murine model of toxoplasmic encephalitis based on the intracerebral inoculation of tachyzoites of the highly virulent RH strain of Toxoplasma gondii. Intracerebral infection resulted in a mortality of 100% on day five. Large inflammatory abscessed infiltrates developed by the second day after inoculation and expanded throughout the remaining period of observation. Numerous tachyzoites were detectable in brain parenchyma by the second day of infection. The marked and precocious histopathological brain lesions produced by the RH strain are useful to detect therapeutic rather than prophylactic effects of drugs against toxoplasmic encephalitis. In this model pyrimethamine, sulphadiazine and its combination were useful in terms of mortality, histopathology and spread of infection whereas clindamycin showed no activity. This murine model of toxoplasmic encephalitis is useful to evaluate antitoxoplasma agents with high therapeutic activity.

An epidemiological study of malaria in Bioko and Annobón islands (Equatorial Guinea).

A seroparasitological study of malaria was carried out in two of the more important islands of Equatorial Guinea, Bioko (ex Fernando Poo) and Annobón. The study involved a randomly-chosen population of children aged from two to nine years. In Bioko 1130 children were chosen from 29 of the 51 villages on the island; in Annobón 185 children were chosen from the capital town, where all the population lived. Indirect immunofluorescence tests (IFAT) showed a prevalence of malaria infection of 29.8% in Bioko and 55.7% in Annobón. The parasitic prevalence (malaria index) was 26.6% and 55.1% respectively, and the splenic index was 57.0% and 54.6% respectively. The results indicate that this is an area of stable hyperendemic malaria, which may benefit from an antimalarial programme.

A comparative pathological study of three strains of Trypanosoma cruzi in an experimental model.

Trypanosoma cruzi, the etiological agent of Chagas' disease, shows a wide variation in its biological behaviour depending on the geographical distribution of different strains. Moreover, some strains can show variations with the course of time. We have studied the tissular tropism of three strains of T. cruzi, Cali, Bolivia and Y, from different geographical origins (Colombia, Bolivia and Brasil respectively) on Swiss mice in order to detect any possible modification in their behaviour attributable only to parasite but not to host variations. The anatomopathological study of sections from heart, brain, liver, spleen, lymphatic ganglion, skeletal muscle and colon from Swiss mice infected with these strains has evidenced the presence of some important discrepancies between the tissular tropism expected from their former descriptions, and classical typification and then observed lesions. The greatest variations were found in the Y strain which had been described as eminently reticulotropic but presented lesions in all the organs except the spleen and lymphatic ganglion. We consider that the variations found in our study can only be explained in terms of changes in the properties of the strains considered, and conclude that the classic typification techniques based on the constancy of the characteristics of the parasite are not fully reliable for the description and clinical management of some evolving strains.

The application of the statistical study in the morphometric characterization of a Colombian strain of Trypanosoma cruzi.

After morphological and histopathological characterization, the Cali strain of T. cruzi isolated from a patient in Cali (Colombia), was also submitted to a morphometrical characterization. For this we estimated 16 cellular parameters in 30 samples of each evolutive form of T. cruzi in blood in NNN medium through planimetry by the IBAS-1 (Image Basic Analytic System). This morphometrical study was completed with a statistical one through the application of the variance (ANOVA-1). This made it possible to evidence a great number of significant differences between the slender, broad and stout forms and also between the short and large epimastigotes from the NNN medium. These data have a real interest for classifying some features of the biological cycle of T. cruzi as well as the future development of diagnosis techniques or therapeutical and prophylactic experiments.