Performance of OptiMAL® in the diagnosis of Plasmodium vivax and Plasmodium falciparum infections in a malaria referral center in Colombia

Alternative, non-microscopic methods for the diagnosis of malaria have recently become available. Among these, rapid dipstick methods stand out. One such test, OptiMAL®, is based on the immunochromatographic detection of Plasmodium lactate dehydrogenase (pLDH) and has the capacity to detect and distinguish infections caused by P. falciparum and Plasmodium sp. This capacity is particularly important in countries where different species of Plasmodium co-exist. In this study we evaluated the performance of OptiMAL® in an urban referral center for malaria diagnosis. Two sets of patients were included: one (n = 112) having predetermined infections with P. falciparum or P. vivax and individuals with negative blood smears; and another consisting of all eligible consecutive patients (n = 80) consulting for diagnosis at the referral center during one month. The overall diagnostic efficiency of OptiMAL® for both sets of patients was 96.9%. Efficiency was higher for P. vivax (98.1%) than for P. falciparum (94.9%). These results corroborate the diagnostic utility of OptiMAL® in settings where P. vivax and P. falciparum co-exist and support its implementation where microscopic diagnosis is unavailable and in circumstances that exceed the capacity of the local microscopic diagnosis facility.

Genetic diversity of Colombian sylvatic Trypanosoma cruzi isolates revealed by the ribosomal DNA

American trypanosomiasis is a common zoonosis in Colombia and Trypanosoma cruzi presents a wide distribution throughout the country. Although some studies based on enzyme electrophoresis profiles have described the population structure of the parasite, very few molecular analyses of genotipic markers have been conducted using Colombian strains. In this study, we amplified the non-transcribed spacer of the mini-gene by PCR, typing the isolates as T. cruzi I, T. cruzi zymodeme 3 or T. rangeli. In addition, the internal transcribed spacers of the ribosomal gene concomitant with the 5.8S rDNA were amplified and submitted to restriction fragment polymorphism analysis. The profiles were analyzed by a numerical methodology generating a phenetic dendrogram that shows heterogeneity among the T. cruzi isolates. This finding suggests a relationship between the complexity of the sylvatic transmission cycle in Colombia and the diversity of the sylvan parasites.

High prevalence anti-Trypanosoma cruzi antibodies, among blood donors in the State of Puebla, a non-endemic area of Mexico

Blood transfusion is the second most common transmission route of Chagas disease in many Latin American countries. In Mexico, the prevalence of Chagas disease and impact of transfusion of Trypanosoma cruzi-contaminated blood is not clear. We determined the seropositivity to T. cruzi in a representative random sample, of 2,140 blood donors (1,423 men and 647 women, aged 19-65 years), from a non-endemic state of almost 5 millions of inhabitants by the indirect hemagglutination (IHA) and enzyme linked immunosorbent assay (ELISA) tests using one autochthonous antigen from T. cruzi parasites, which were genetically characterized like TBAR/ME/1997/RyC-V1 (T. cruzi I) isolated from a Triatoma barberi specimen collected in the same locality. The seropositivity was up to 8.5% and 9% with IHA and ELISA tests, respectively, and up to 7.7% using both tests in common. We found high seroprevalence in a non-endemic area of Mexico, comparable to endemic countries where the disease occurs, e.g. Brazil (0.7%), Bolivia (13.7%) and Argentina (3.5%). The highest values observed in samples from urban areas, associated to continuous rural emigration and the absence of control in blood donors, suggest unsuspected high risk of transmission of T. cruzi, higher than those reported for infections by blood e.g. hepatitis (0.1%) and AIDS (0.1%) in the same region.

Biological characterization of a beta-galactosidase expressing clone of Trypanosoma cruzi CL strain

Clone CL B5 of Trypanosoma cruzi is a beta-galactosidase expressing organism that was genetically transfected to be used for in vitro pharmacological screening. Biological parameters were determined, evaluating growth kinetics of epimastigotes, metacyclogenesis, infectivity to mammalian cell lines, parasitemia kinetics in mice and sensibility to nifurtimox and benznidazole. Differences in relation to other strains and CL parental strain were found, the most important being the incapability to produce death to mice in spite of the high inoculum used. However, it possesses the required features to be used for in vitro drug screening. Data obtained demonstrate that heterogeneity of T. cruzi appears even among clones of the same strain, and that these differences found do not prevent the use of clone CL B5 for the purpose that was engineered.

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37°C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.

Should Trypanosoma cruzi be called "cruzi" complex? A review of the parasite diversity and the potential of selecting population after in Vitro culturing and mice infection

Morpho-biological diversity of Trypanosoma cruzi has been known since Chagas' first works in 1909. Several further studies confirmed the morphological differences among the parasite strains, which were isolated from different reservoirs and vectors, as well as from human beings. In the early sixties, antigenic differences were found in the parasite strains from various sources. These differences, coupled to the observation of regional variations of the disease, led to the proposal of the term cruzi complex to designate the taxon T. cruzi. Since then this protozoan has been typed in distinct biodemes, zymodemes and lineages which were consensually grouped into T. cruzi I, T. cruzi II and into non-grouped strains. T. cruzi genotypic characterization, initially carried out by schizodeme analysis and more recently by various other techniques, has shown a great diversity of the parasite strains. In fact, T. cruzi is formed by groups of heterogeneous sub-population, which present specific characteristics, including distinct histotropism. The interaction of the different infecting clones of the cruzi complex and the human host will determine the morbidity of the disease.

Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37°C. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same.

A novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi

We report the molecular characterization of a novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi. Steady-state level of transcripts of this sequence family appeared to be developmentally regulated, since only in the replicative forms the parasite showed expression of related sequences with a major band around 3 kb. The presence of frame shifts or premature stop codons predicts that transcripts are not translated. The sequence family also contains truncated forms of retrotransposons elements that may become potential hot spots for retroelement insertion. Sequences homologous to this family are interspersed at many chromosomes including the subtelomeric regions.

In vitro evaluation of the activity of aromatic nitrocompounds against Trypanosoma cruzi

Fourteen compounds were evaluated for their activity against Trypanosoma cruzi blood stream forms at the concentration of 500 µg/ml. Six compounds were active and re-tested at lower concentrations.

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.

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.

Epidemiology of Chagas disease in Guatemala: infection rate of Triatoma dimidiata, Triatoma nitida and Rhodnius prolixus (Hemiptera, Reduviidae) with Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida, Trypanosomatidae)

A five-year domiciliary collection in the 22 departments of Guatemala showed that out of 4,128 triatomines collected, 1,675 were Triatoma dimidiata (Latreille, 1811), 2,344 were Rhodnius prolixus Stal 1859, and only 109 were T. nitida Usinger 1939. The Chagas disease parasite, Trypanosoma cruzi, was found in all three species. Their natural infection rates were similar in the first two species (20.6%; 19.1%) and slightly lower in T. nitida(13.8%). However there was no significant difference in the infection rates in the three species (p = 0.131). T. dimidiata males have higher infection rates than females (p = 0.030), whereas for R. prolixus there is no difference in infection rates between males and females (p = 0.114). The sex ratios for all three species were significantly skewed. More males than females were found inside houses for T. dimidiata (p < 0.0001) and T. nitida (p = 0.011); a different pattern was seen for R. prolixus (p = 0.037) where more females were found. Sex ratio is proposed as an index to show the mobility of T. dimidiata in different populations. T. dimidiata is widely distributed in the country, and is also the main vector in at least ten departments, but R. prolixus with higher vectorial capacity is an important vector in at least two departments.

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.

Diterpenoids from Azorella compacta (Umbelliferae) active on Trypanosoma cruzi

The anti-Trypanosoma cruzi activity of natural products isolated from Azorella compacta was evaluated, with particular emphasis on their effect against intracellular amastigotes. Five diterpenoids from A. compacta derived from mulinane and azorellane were isolated and identified. Only two products, named azorellanol (Y-2) and mulin-11,3-dien-20-oic acid (Y-5), showed trypanocidal activity against all stages of T. cruzi including intracellular amastigotes. At 10 µM, these compounds displayed a strong lytic activity. It ranged from 88.4 ± 0.6 to 99.0 ± 1 % for all strains and stages evaluate, with an IC50 /18 h values of 20-84 µM and 41-87 µM, respectively. The development of intracellular amastigotes was also inhibited by nearly 60% at 25 µM. The trypanocidal molecules Y-2 and Y-5 did show different degrees of cytotoxicity depending on the cell line tested, with an IC50 /24 h ranging from 33.2 to 161.2 µM. We evaluated the effect of diterpenoids against intracellular T. cruzi forms by immunofluorescent identification of a specific membrane molecular marker (Ssp-4 antigen) of the T. cruzi amastigote forms. The accuracy and reproducibility of the measurements were found to be outstanding when examined by confocal microscopy.

Trypanosoma (Herpetosoma) rangeli Tejera, 1920: an updated review

Trypanosoma rangeli, a parasite generally considered non-pathogenic for man, is the second species of human trypanosome to be reported from the New World. The geographical distribution of T. rangeli often overlaps with that of T. cruzi, the same vertebrate and invertebrate hosts being infected. Their differentiation thus becomes of real, practical importance, particularly as they share approximately half the antigenic determinants recognized by the humoral response. Little is known about the life cycle of T. rangeli in the vertebrate host, although thousands of human and wild animal infections have been reported. Recent studies have revealed 2 major phylogenetic lineages in T. rangeli having different characteristics, thus leading to better understanding of the epidemiology and interactions with this parasite's vertebrate hosts and triatomine vectors. Based on further genetic characterization analysis, the authors have proposed 2 alternative hypotheses and consider that T. rangeli could have had clonal evolution or have been subjected to speciation processes.