Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme

Parasites belonging to Leishmania braziliensis, Leishmania donovani, Leishmania mexicana complexes and Trypanosoma cruzi (clones 20 and 39) were searched in blood, lesions and strains collected from 28 patients with active cutaneous leishmaniasis and one patient with visceral leishmaniasis. PCR-hybridization with specific probes of Leishmania complexes (L. braziliensis, L. donovani and L. mexicana) and T. cruzi clones was applied to the different DNA samples. Over 29 patients, 8 (27.6%) presented a mixed infection Leishmania complex species, 17 (58.6%) a mixed infection Leishmania-T. cruzi, and 4 (13.8%) a multi Leishmania-T. cruzi infection. Several patients were infected by the two Bolivian major clones 20 and 39 of T. cruzi (44.8%). The L. braziliensis complex was more frequently detected in lesions than in blood and a reverse result was observed for L. mexicana complex. The polymerase chain reaction-hybridization design offers new arguments supporting the idea of an underestimated rate of visceral leishmanisis in Bolivia. Parasites were isolated by culture from the blood of two patients and lesions of 10 patients. The UPGMA (unweighted pair-group method with arithmetic averages) dendrogram computed from Jaccard's distances obtained from 11 isoenzyme loci data confirmed the presence of the three Leishmania complexes and undoubtedly identified human infections by L. (V.) braziliensis, L. (L.) chagasi and L. (L.) mexicana species. Additional evidence of parasite mixtures was visualized through mixed isoenzyme profiles, L. (V.) braziliensis-L. (L.) mexicana and Leishmania spp.-T. cruzi.The epidemiological profile in the studied area appeared more complex than currently known. This is the first report of parasitological evidence of Bolivian patients with trypanosomatidae multi infections and consequences on the diseases' control and patient treatments are discussed.

Impact of residual spraying on Rhodnius prolixus and Triatoma dimidiata in the department of Zacapa in Guatemala

As a vector control program to control Chagas disease in Guatemala, residual spraying of Rhodnius prolixus and Triatoma dimidiata was performed, and its impact was measured in the department of Zacapa. In order to identify infested villages and determine the degree of infestation, a baseline entomological survey to identify municipalities infested with vectors followed by an additional vector survey in areas known to be infested was conducted. Residual spraying using pyrethroid insecticides was performed at all the villages identified as being infested with the vectors. The residual spraying was shown to be highly effective against both vectors by the decrease in infestation indices after spraying. Analysis of the cost-effectiveness of the spraying showed that the average cost of insecticides per house is high when compared with that in Southern Cone countries.

Trypanosoma cruzi-elicited CD8+ T cell-mediated myocarditis: chemokine receptors and adhesion molecules as potential therapeutic targets to control chronic inflammation?

In Chagas disease, during the acute phase, the establishment of inflammatory processes is crucial for Trypanosoma cruzi control in target tissues and for the establishment of host/parasite equilibrium. However, in about 30% of the patients, inflammation becomes progressive, resulting in chronic disease, mainly characterized by myocarditis. Although several hypothesis have been raised to explain the pathogenesis of chagasic myocardiopathy, including the persistence of the parasite and/or participation of autoimmune processes, the molecular mechanisms underlying the establishment of the inflammatory process leading to parasitism control but also contributing to the maintenance of T. cruzi-elicited chronic myocarditis remain unsolved. Trying to shed light on these questions, we have for several years been working with murine models for Chagas disease that reproduce the acute self-resolving meningoencephalitis, the encephalitis resulting of reactivation described in immunodeficient individuals, and several aspects of the acute and chronic myocarditis. In the present review, our results are summarized and discussed under the light of the current literature. Furthermore, rational therapeutic intervention strategies based on integrin-mediated adhesion and chemokine receptor-driven recruitment of leukocytes are proposed to control T. cruzi-elicited unbalanced inflammation.

Comparative study of the stridulatory sulcus, buccula and rostrum of the nymphs of Triatoma guazu Lent & Wygodzinsky, 1979 and Triatoma jurbergi Carcavallo, Galvão & Lent, 1998 by scanning electron microscopy (Hemiptera, Reduviidae)

The ultrastructural morphology of the ventral region of the head (rostrum and buccula) and proesternum (stridulatory sulcus) of nymphs from the 1st to 5th instars of Triatoma guazu Lent & Wygodzinsky, 1979 and Triatoma jurbergi Carcavallo, Galvão & Lent, 1998 was described. Morphological differences between the two species and of the five nymphal stages development of each species were observed. These structures showed systematic differential characteristics of the studied species and may be used to increase their taxonomic range.

Triatoma rubrovaria (Blanchard, 1843) (Hemiptera-Reduviidae-Triatominae) III: patterns of feeding, defecation and resistance to starvation

Data from the Chagas Disease Control Program indicate a growing domiciliary and peridomiciliary invasion of Triatoma rubrovaria in the State of Rio Grande do Sul, where it has become the most frequent triatomine species captured there since the control of T. infestans. Bionomic characteristics that could influence the vectorial capacity of T. rubrovaria as vector of Trypanosoma cruzi were evaluated: patterns of (i) feeding, (ii) defecation, and (iii) resistance to starvation, using insects fed on mice. Fifty three percent of the females showed a defecation pattern conducive to chagasic transmission, defecating either on or near the bite site. The averages of the resistance to starvation varied from 48.1 to 179 days, for the first and fifth nymphal stages, respectively. Our study shows that with respect to the patterns of feeding, defecation and resistance to fasting, T. rubrovaria presented similar rates to the ones observed for other effective vector species, such as T. infestans. Thus, based on our studies we conclude that T. rubrovaria has biological characteristics that can positively influence its capacity to become infected and transmit T. cruzi, and also to keep residual populations after chemical control interventions.

Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ polymerase chain reaction

The use of in situ techniques to detect DNA and RNA sequences has proven to be an invaluable technique with paraffin-embedded tissue. Advances in non-radioactive detection systems have further made these procedures shorter and safer. We report the detection of Trypanosoma cruzi, the causative agent of Chagas disease, via indirect and direct in situ polymerace chain reaction within paraffin-embedded murine cardiac tissue sections. The presence of three T. cruzi specific DNA sequences were evaluated: a 122 base pair (bp) sequence localized within the minicircle network, a 188 bp satellite nuclear repetitive sequence and a 177 bp sequence that codes for a flagellar protein. In situ hybridization alone was sensitive enough to detect all three T. cruzi specific DNA sequences.

Characterization of [Ca2+]i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes

Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1% while A23187 (0.5 µM) alone did not induce significant effects (17%); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC.

Genitourinary changes in hamsters infected and reinfected with Trypanosoma cruzi

Authors describe genitourinary changes in male hamsters infected and reinfected with Trypanosoma cruzi. Changes in genital organs have been described in human and in experimental chagasic infection. Genital dysfunctions in chronic chagasic patients affect ejaculation, libido and sexual potency, and testis biopsies may show arrested maturation of germ cells, oligozoospermia and azoospermia. Sixty-five male hamsters were inoculated and reinoculated with 2x10³ trypomastigotes of T. cruzi VIC strain, and 22 non-infected animals constituted the control group. Animals were necropsied and fragments from testis, epididymis, seminal vesicle and bladder were collected and stained with hematoxylin-eosin. Peroxidase anti-peroxidase procedure was utilized to detect tissue parasitism. T. cruzi nests were found in testis, epididymis and seminal vesicle of these hamsters. Such parasitism plays a role in the origin of genital lesions observed in humans and laboratory animals during chronic chagasic infection.

Action of Trypanosoma rangeli in infections with virulent Trypanosoma cruzi populations

In experimental murine infections with Trypanosoma rangeli it has been observed development immune response to Trypanosoma cruzi. The aim of the present work was to analyze the result of antigenic stimuli and the protective effect with T. rangeli in T. cruzi infections. Mice groups immunized with metacyclic trypomastigotes of T. rangeli (Choachí-2V strain), derived from haemolymph and salivary gland and reinfected with T. cruzi virulent populations (Tulahuen strain, SA strain and Dm28c clone) from infected in vitro cells, showed decrease severity of disease outcomes, low parasitemia levels and 100% survival of all mice immunized, in comparison with groups infected only with T. cruzi populations, which demonstrated tissue affection, high parasitemia levels and the death of all animals. The above mentioned data contribute to understand the biological behaviour of T. cruzi and T. rangeli and their interaction with vertebrate host.

8-Methoxy-naphtho[2,3-b]thiophen-4,9-quinone, a non-competitive inhibitor of trypanothione reductase

The enzyme trypanothione reductase is a recognised drug target in trypanosomatids and has been used in the search of new compounds with potential activity against diseases such as leishmaniasis, Chagas disease and African trypanosomiasis. 8-Methoxy-naphtho [2,3-b] thiophen-4,9-quinone was selected in a screening of natural and synthetic compounds using an in vitro assay with the recombinant enzyme from Trypanosoma cruzi. Its mode of inhibition fits a non-competitive model with respect to the substrate (trypanothione) and to the co-factor (NADPH), with Ki-values of 5 and 3.6 µM, respectively. When tested against human glutathione reductase, this compound did not display any significant inhibition at 100 µM, indicating a good selectivity against the parasite enzyme.

Electrocardiographic findings in Mexican chagasic subjects living in high and low endemic regions of Trypanosoma cruzi infection

In México the first human chronic chagasic case was recognized in 1940. In spite of an increasing number of cases detected since that time, Chagas disease in México has been poorly documented. In the present work we studied 617 volunteers subjects living in high and low endemic regions of Trypanosoma cruzi infection with seroprevalence of 22% and 4% respectively. Hemoculture performed in those seropositive subjects failed to demonstrate circulating parasites, however polymerase chain reaction identified up to 60% of them as positives. A higher level of anti-T. cruzi antibodies was observed in seropositive residents in high endemic region, in spite of similar parasite persistence (p < 0.05). On standard 12 leads electrocardiogram (ECG) 20% to 22% seropositive individuals from either region showed right bundle branch block or ventricular extrasystoles which were more prevalent in seropositive than in seronegative individuals (p < 0.05). In conclusion, the frequency or type of ECG abnormality was influenced by serologic status but not by endemicity or parasite persistence. Furthermore, Mexican indeterminate patients have a similar ECG pattern to those reported in South America.

Estudo morfométrico do gênero Panstrongylus Berg, 1879 (Hemiptera, Reduviidae, Triatominae)

The subfamily Triatominae is actually represented by 137 species distributed among 6 tribes and 19 genera. Within this subfamily, the genus Panstrongylus, Berg 1879, is composed by 13 species widespread in sylvatic, peridomestic, and domestic habitats of Neotropical regions. These species are vectors of Chagas disease and consequently are found associated with its main hosts, such as birds and mammals. Interest in species of this genus has been increasing in the last few years. Reports of several authors indicate these Triatominae to invade and colonize houses, increasing their epidemiological significance. Morphometry was used in this study to investigate correlations among possible closely related species. We measured 224 specimens among 13 species through a set of metric variables of the head. The results indicated that the genus Panstrongylus seems to be homogeneous since 10 of the 14 species were shown to be closely related.

Comparative evaluation of pyrethroid insecticide formulations against Triatoma infestans (Klug): residual efficacy on four substrates

We investigated the residual efficacy of four insecticide formulations used in Chagas disease vector control campaigns: cyfluthrin 12.5% suspension concentrace (SC), lambda-cyhalothrin 10% wettable powder (WP), deltamethrin 2.5% SC, and 2.5% WP on four types of circular blocks of wood, straw with mud, straw with mud painted with lime, and mud containing 5% of cement. Three concentrations of these insecticides were tested: the LC90 (previously determined on filter paper), the double of the LC90, and the recommended operational dose. For each bioassay test, 15 third-stage nymphs of Triatoma infestans (Klug) (Hemiptera: Reduviidae) were exposed for 120 h to each treatment at 24 h, 30, 60, 90, and 180 days post-spraying. Mortality rates, moulting history and behaviour were recorded at 24, 48, 72, and 120 h of exposure. Mortality rates were highest during the first 30 days post-spraying. Highest mortality rates (above 50%) were observed for deltamethrin 2.5% SC and lambda-cyhalothrin 10% WP on wood blocks up to three months post-spraying. Mud was the substrate on which treatments showed lowest persistence, with the other two substrates showing intermediate residual efficacy of all treatments. During the first 30 days WP formulations were not as effective as SC flowable formulations but, overall in the longer term, WP gave grater mortality rates of T. infestans nymphs exposed at up to six months post-spraying. Porous surfaces, especially mud, showed most variability presumably due to absorption of the insecticide. In contrast the less porous surfaces (i.e. wood and lime-coated mud) kept mortality rates high for longer post-treatment, irrespective of the insecticide concentration used.

Preliminary results of random amplification of polymorphic DNA among Triatominae of the phyllosoma complex (Hemiptera, Reduviidae)

In Mexico, Triatoma longipennis (Usinger), Triatoma picturata (Usinger), and Triatoma pallidipennis (Stal), primary Chagas disease vector species of the phyllosoma complex, were analyzed by randomly amplified polymorphic DNA (RAPD). Sixteen decametric primers resolved individual profiles not identical, but partially discriminative between species. Analysis based on pairwise presence/absence comparisons between the three species was performed using three primers and two outgroup species Triatoma infestans (Klug) and Triatoma barberi (Usinger). Fifty-three bands in total were scored, although only two bands were constant among the three phyllosoma complex species. Two other bands were constant only for T. longipennis and T. picturata together, and not present in T. pallidipennis. Neighbor Joining tree and the multiple correspondence analysis discriminated T. pallidipennis clearly from the other two species, although there was overlap between T. longipennis and T. picturata. The results indicate a close relationship between the studied species and support the hypothesis of their recent evolution. The suitability of RAPD to discern populations within the species is discussed.

Changes associated with laboratory rearing in antennal sensilla patterns of Triatoma infestans, Rhodnius prolixus, and Rhodnius pallescens (Hemiptera, Reduviidae, Triatominae)

We examined changes in the array of antennal sensilla of three species of Triatominae (Triatoma infestans, Rhodnius prolixus, and R. pallescens) following their establishment for different periods in laboratory culture. In each case, the laboratory colonies were compared with conspecific samples taken directly from the field, by quantitative analysis of the sensilla arrays on the three distal segments of the antenna in terms of the densities of three types of chemoreceptors (basiconics and thick and thin walled trichoids) and one type of mechanoreceptor (bristles). Sensilla densities were compared by ANOVA or non-parametric tests, and by multivariate discriminant analysis. Strains of the same species reared in different laboratories showed significant differences in their sensilla arrays, especially when compared to field-collected material from the same geographic origin. A Bolivian strain of T. infestans reared in the laboratory for 15 years and fed at monthly intervals, showed greatest differences from its conspecific wild forms, especially in terms of reductions in the number of chemoreceptors. By contrast, an Argentine strain of T. infestans reared for 25 years in the laboratory and fed weekly, showed a relative increase in the density of mechanoreceptors. A Colombian strain of R. prolixus reared for 20 years and fed weekly or fortnightly, showed only modest differences in the sensilla array when compared to its wild populations from the same area. However, a Colombian strain of R. pallescens reared for 12 years and fed fortnightly, did show highly significant reductions in one form of chemoreceptor compared to its conspecific wild populations. For all populations, multivariate analysis clearly discriminated between laboratory and field collected specimens, suggesting that artificial rearing can lead to modifications in the sensory array. This not only supports the idea of morphological plasticity in these species, but also suggests caution in the use of long-established laboratory material for experimental studies designed to extrapolate the natural behaviour and physiology of these species.