Ana María Mejía-Jaramillo

Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced-leader genes.

The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harbouring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia+Tc Id, Tc Ia+Tc Ie and Tc Id+Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time.

Trypanosoma cruzi: Biological characterization of lineages I and II supports the predominance of lineage I in Colombia.

The causes of the particular distribution of both Trypanosoma cruzi lineages throughout the American continent remain unknown. In Colombia, T. cruzi I is the predominant group in both domestic and sylvatic cycles. Here, we present the biological characterization of T. cruzi parasites belonging to both T. cruzi I and T. cruzi IIb groups. Our results show the inability of the T. cruzi IIb clones to infect mammalian cells, produce trypomastigotes and replicate in Rhodnius prolixus, the main vector species in this country. Moreover, this result was confirmed when other species from the same genus, such as R. pallescens and R. robustus, were infected with the same TcIIb clone and its parental strain, while the infection in other genera such as Triatoma and Panstrongylus was successful. Furthermore, the growth kinetics and duplication time in vitro suggest that the high prevalence of T. cruzi I in Colombia results from more successful interactions between parasite lineage, vector, and host species. This type of study may help to understand the factors influencing the particular epidemiological patterns of Chagas disease transmission in different endemic regions.

Biological characterization of Trypanosoma cruzi stocks from domestic and sylvatic vectors in Sierra Nevada of Santa Marta, Colombia.

Sierra Nevada of Santa Marta is one of the most endemic regions of Chagas disease in Colombia. In this study, we compared the biological behavior and genetic features of Trypanosoma cruzi stocks that were isolated from domestic and sylvatic insects in this area. Rhodnius prolixus (from domestic environments) and Triatoma dimidiata (from sylvatic, peridomestic and domestic environments) are the most important vectors in this region. Genetic characterization showed that all stocks corresponded to T. cruzi I, but LSSP-PCR analyses indicated that some genotypes were present in both environments. Biological characterization in vitro showed a low growth rate in sylvatic T. cruzi stocks and in some domestic T. cruzi stocks, possibly indicating the presence of stocks with similar behavior in both transmission cycles. In parallel, in vivo behavioral analysis also indicated that T. cruzi stocks are variable and this species did not show a correlation between the environments where they were isolated. In addition, all stocks demonstrated a low mortality rate and histopathological lesions in heart, skeletal muscle and colon tissue. Moreover, our data indicated that experimentally infected chagasic mice displayed a relation between their myocardial inflammation intensity, parasitism tissue and parasite load using the qPCR. In conclusion, our results indicate that the T. cruzi stocks present in SNSM have similar biological behavior and do not show a correlation with the different transmission cycles. This could be explained by the complex transmission dynamics of T. cruzi in Sierra Nevada of Santa Marta, where hosts, vectors (e.g., T. dimidiata) and reservoirs circulate in both environments due to the close contact between the two transmission cycles, favoring environment overlapping. This knowledge is an important key to understanding the epidemiology and pathology of Chagas disease in this Colombian region. Furthermore, our findings could be of significant use in the design of control strategies restricted to a specified endemic region.

High-Resolution Melting (HRM) of the Cytochrome B Gene: A Powerful Approach to Identify Blood-Meal Sources in Chagas Disease Vectors

Methods to determine blood-meal sources of hematophagous Triatominae bugs (Chagas disease vectors) are serological or based on PCR employing species-specific primers or heteroduplex analysis, but these are expensive, inaccurate, or problematic when the insect has fed on more than one species. To solve those problems, we developed a technique based on HRM analysis of the mitochondrial gene cytochrome B (Cyt b). This technique recognized 14 species involved in several ecoepidemiological cycles of the transmission of Trypanosoma cruzi and it was suitable with DNA extracted from intestinal content and feces 30 days after feeding, revealing a resolution power that can display mixed feedings. Field samples were analyzed showing blood meal sources corresponding to domestic, peridomiciliary and sylvatic cycles. The technique only requires a single pair of primers that amplify the Cyt b gene in vertebrates and no other standardization, making it quick, easy, relatively inexpensive, and highly accurate.

Geographical clustering of Trypanosoma cruzi I groups from Colombia revealed by low-stringency single specific primer-PCR of the intergenic regions of spliced-leader genes.

A low-stringency single-primer polymerase chain reaction (LSSP-PCR) typing procedure targeted to the intergenic regions of spliced-leader genes (SL) was designed to profile Trypanosoma cruzi I stocks from endemic regions of Colombia. Comparison between SL-LSSP-PCR profiles of parasite DNA from vector faeces and cultures isolated from those faeces showed more conservative signatures than profiles using LSSP-PCR targeted to the minicircle variable regions (kDNA). This was also observed by analysing 15 parasite clones from one stock as well as serial samples of a same stock after in vitro culturing or inoculation into mice. Thus, SL-LSSP-PCR appears more appropriate than kDNA-LSSP-PCR for reliable typing of major T. cruzi I groups from in vitro cultured stocks and triatomine faeces. SL-LSSP-PCR grouped 46 of 47 T. cruzi I Colombian stocks according to their geographical procedences in four clusters: Cluster Cas from Casanare Department, Cluster Mg from Northern Magdalena department, Cluster Mom from Momposina Depression in Southern Magdalena and finally Cluster NW from northwestern Colombia, including Sucre, Chocó, Córdoba and Antioquia departments. Sequence analysis identified punctual mutations among amplicons from each cluster. Within Cluster Mg, sequence polymorphism allowed association with different sylvatic vector species. Novel SL sequences and LSSP-PCR profiles are reported from T. cruzi I infecting Eratyrus cuspidatus, Panstrongylus geniculatus and Rhodnius pallescens vectors.

Gene expression study using real-time PCR identifies an NTR gene as a major marker of resistance to benznidazole in Trypanosoma cruzi

BackgroundChagas disease is a neglected illness, with limited treatments, caused by the parasite Trypanosoma cruzi. Two drugs are prescribed to treat the disease, nifurtimox and benznidazole, which have been previously reported to have limited efficacy and the appearance of resistance by T. cruzi. Acquisition of drug-resistant phenotypes is a complex physiological process based on single or multiple changes of the genes involved, probably in its mechanisms of action.ResultsThe differential genes expression of a sensitive Trypanosoma cruzi strain and its induced in vitro benznidazole-resistant phenotypes was studied. The stepwise increasing concentration of BZ in the parental strain generated five different resistant populations assessed by the IC50 ranging from 10.49 to 93.7 μM. The resistant populations maintained their phenotype when the BZ was depleted from the culture for many passages. Additionally, the benznidazole-resistant phenotypes presented a cross-resistance to nifurtimox but not to G418 sulfate. On the other hand, four of the five phenotypes resistant to different concentrations of drugs had different expression levels for the 12 genes evaluated by real-time PCR. However, in the most resistant phenotype (TcR5x), the levels of mRNA from these 12 genes and seven more were similar to the parental strain but not for NTR and OYE genes, which were down-regulated and over-expressed, respectively. The number of copies for these two genes was evaluated for the parental strain and the TcR5x phenotype, revealing that the NTR gene had lost a copy in this last phenotype. No changes were found in the enzyme activity of CPR and SOD in the most resistant population. Finally, there was no variability of genetic profiles among all the parasite populations evaluated by performing low-stringency single-specific primer PCR (LSSP-PCR) and random amplified polymorphic DNA RAPD techniques, indicating that no clonal selection or drastic genetic changes had occurred for the exposure to BZ.ConclusionHere, we propose NTR as the major marker of the appearance of resistance to BZ.

Genotyping of Trypanosoma cruzi in a hyper-endemic area of Colombia reveals an overlap among domestic and sylvatic cycles of Chagas disease

BackgroundChagas disease is a neglected illness caused by the Trypanosoma cruzi parasite, which widely affects American communities. This study attempted to identify T. cruzi genotypes circulating in four indigenous communities of the Sierra Nevada of Santa Marta, Colombia, to investigate parasite transmission dynamics in these communities. In addition, some epidemiological variables to determine the risk factors for infection with this parasite, such as the prevalence of T. cruzi infection, the triatomine species, and the domestic and sylvatic mammals that act as vectors and reservoirs of the parasite in the domestic, peridomestic and sylvatic cycles, were examined.MethodsWe developed a prospective study to identify the main risk factors associated with T. cruzi infection in the region. The T. cruzi prevalence was determined by ELISA, IFA and PCR. Triatomines species and both domestic and sylvatic mammals from all communities were captured and sampled. To analyze parasite transmission dynamics in these four communities, eight DNA parasite probes were generated from insect and reservoir samples, and a DNA blot analysis were carried out.ResultsSerological studies revealed 37% prevalence in the four communities, and Kasakumake was the most endemic region, containing approximately 70% seropositives. Moreover, the molecular diagnosis showed a high correlation between the serological data and the T. cruzi circulating in the patients’ blood. A total of 464 triatomine insects were collected in domestic, peridomestic and sylvatic environments, and these insects belonged to five different species; Rhodnius prolixus and Triatoma dimidiata were the two more important species transmitting the parasite. After studying the eco-epidemiological factors in these four communities, the most important risk factors for infection with the parasite were determined. These risk factors are a high infection rate of people and domestic animals, the construction materials of the houses, the presence of infected triatomines inside the human dwellings, the proximity between houses and a sylvatic environment with several triatomine species and wild animals. Finally, the molecular characterization of T. cruzi showed the presence of three haplotypes and complex T. cruzi mixed infections in all reservoirs.ConclusionsActive transmission of T. cruzi is present in four indigenous communities of the Sierra Nevada de Santa Marta with overlap between the domestic and the sylvatic transmission cycles of Chagas disease.

Infection Rates by Dengue Virus in Mosquitoes and the Influence of Temperature May Be Related to Different Endemicity Patterns in Three Colombian Cities

Colombia is an endemic country for dengue fever where the four serotypes of virus dengue (DENV1–4) circulate simultaneously, and all types are responsible for dengue cases in the country. The control strategies are guided by entomological surveillance. However, heterogeneity in aedic indices is not well correlated with the incidence of the disease in cities such as Riohacha, Bello and Villavicencio. As an alternative, molecular detection of dengue virus in mosquitoes has been proposed as a useful tool for epidemiological surveillance and identification of serotypes circulating in field. We conducted a spatiotemporal fieldwork in these cities to capture adult mosquitoes to assess vector infection and explain the differences between Breteau indices and disease incidence. DENV infection in females and DENV serotype identification were evaluated and infection rates (IR) were estimated. The relationship between density, dengue cases and vector index was also estimated with logistic regression modeling and Pearson’s correlation coefficient. The lack of association between aedic indices and dengue incidence is in agreement with the weak associations between the density of the mosquitoes and their infection with DENV in the three cities. However, association was evident between the IR and dengue cases in Villavicencio. Furthermore, we found important negative associations between temperature and lag time from two to six weeks in Riohacha. We conclude that density of mosquitoes is not a good predictor of dengue cases. Instead, IR and temperature might explain better such heterogeneity.

Trypanosoma cruzi strains resistant to benznidazole occurring in Colombia

INTRODUCTION Traditional medicine is an invaluable source of research into new medicines as a supplement for the treatment of snakebite, considered as a serious public health problem worldwide. The extracts of the medicinal plant, Renealmia alpina, have been used traditionally by indigenous people of Chocó (Colombia) against Bothrops asper snakebite, a snake responsible for the majority of snakebite accidents in Colombia. OBJECTIVE The ability of extracts of R. alpinia leaves was tested for its ability to neutralize the hemorrhagic, coagulant and proteolytic effects of the snakebite venom of B. asper. MATERIALS AND METHODS The acute toxicity tests and analgesic activity of R. alpina were evaluated in vivo. In addition, tests were undertaken in in vitro conditions to demonstrate inhibition of coagulant, haemolytic and proteolytic activity of the B. asper venom. Results. Renealmia alpinia extracts had no toxic effects in experimental animals and also provided analgesic and antiophidian effects and protection against the lethal effects of the venom of B. asper. CONCLUSION Renealmia. alpinia was an effective therapeutic alternative in association with antivenom treatment in the event of a B. asper snakebite accident. It was demonstrated to protect against the lethal effects and provided analgesic properties as well.

Parasitological and molecular surveys reveal high rates of infection with vector-borne pathogens and clinical anemia signs associated with infection in cattle from two important livestock areas in Colombia

In Colombia, vector-borne diseases are one of the most important problems in the livestock industry. The present study reports parasitological and molecular surveys of vector-borne pathogens in cattle from two high-value livestock areas in Colombia. A total of 464 samples (226 from Antioquia and 238 from Arauca) were analyzed. While the blood smear analysis identified 98 (21.1%), 14 (3.0%) and 30 (6.5%) positive samples for Anaplasma spp., Babesia spp. and Trypanosoma spp., respectively, the molecular methods indicated that 275 (59.3%), 146 (31.5%), 64 (13.8%), 236 (50.9%) and 43 (9.3%) of the samples were positive for Anaplasma marginale, Babesia bigemina, Babesia bovis, Trypanosoma theileri and T. evansi, respectively. Mixed infections were detected in 250 (53.9%) samples. Interestingly, animals aged ≤1 year had higher probabilities of being infected with A. marginale and Babesia spp., and lower probabilities of being infected with Trypanosoma spp., while the animals raised under intensive system breeding had higher probabilities of being infected with all pathogens. Additionally, T. theileri infection was found in higher prevalence in anemic animals than animals with normal packed cell volume (PCV). This is the first molecular report that evaluated the infection with three genders of vector-borne pathogens in cattle in Colombia and provides useful information for a better understanding of the epidemiologic aspects, as well as for the management and control, of these diseases.