Chagas disease, caused by Trypanosoma cruzi (T. cruzi), is a serious public health problem. Current treatment is restricted to two drugs, benznidazole and nifurtimox, displaying serious efficacy and ...safety drawbacks. Nucleoside analogues represent a promising alternative as protozoans do not biosynthesize purines and rely on purine salvage from the hosts. Protozoan transporters often present different substrate specificities from mammalian transporters, justifying the exploration of nucleoside analogues as therapeutic agents. Previous reports identified nucleosides with potent trypanocidal activity; therefore, two 7-derivatized tubercidins (FH11706, FH10714) and a 3′-deoxytubercidin (FH8513) were assayed against T. cruzi. They were highly potent and selective, and the uptake of the tubercidin analogues appeared to be mediated by the nucleoside transporter TcrNT2. At 10 μM, the analogues reduced parasitemia >90% in 2D and 3D cardiac cultures. The washout assays showed that FH10714 sterilized the infected cultures. Given orally, the compounds did not induce noticeable mouse toxicity (50 mg/kg), suppressed the parasitemia of T. cruzi-infected Swiss mice (25 mg/kg, 5 days) and presented DNA amplification below the limit of detection. These findings justify further studies with longer treatment regimens, as well as evaluations in combination with nitro drugs, aiming to identify more effective and safer therapies for Chagas disease.
In the Amazon region, several species of triatomines occur in the natural environments. Among them, species of the genus
are a risk to human populations due to their high rates of infection with
The ...aim of this study was to identify the
genotypes in
specimens and their relationship with sylvatic hosts from different environments in the Brazilian Amazon.
A total of 492 triatomines were collected from the municipalities of Monte Negro, Rondônia state, and Humaitá, Amazonas state, 382 of them being nymphs and 110 adults. Genotyping of
in six discrete typing units (DTUs) was performed using conventional multilocus PCR. The triatomines that were positive for
and engorged with blood were also targeted for amplification of the
gene to identify bloodmeal sources.
Of the 162 positive samples, the identified DTUs were TcI (87.65%) and TcIV (12.35%). It was observed that 102 specimens were engorged with a variety of bloodmeals. Triatomines infected with TcI were associated with DNA of all identified vertebrates, except
TcIV was detected in triatomines that fed on
,
,
,
,
,
, and
Results highlight the need to understand the patterns of
genotypes in
spp. and their association with sylvatic hosts to better elucidate their role in the transmission of Chagas disease in the Amazon region.
Currently, approximately 70% of new cases of Chagas disease (CD) in Brazil are attributed to oral transmission, particularly through foods such as açaí, bacaba, and sugarcane juice, primarily in the ...northern and northeastern regions of the country. This underscores the imperative need to control the spread of the disease. The methods utilized to conduct quality control for food associated with outbreaks and to assess the potential for the oral transmission of CD through consuming açaí primarily rely on isolating the parasite or inoculating food into experimental animals, restricting the analyses to major research centers. While there are existing studies in the literature on the detection and quantification of
DNA in açaí, the evaluation of parasites' viability using molecular methods in this type of sample and differentiating between live and dead parasites in açaí pulp remain challenging. Consequently, we developed a molecular methodology based on RT-qPCR for detecting and quantifying viable
in açaí pulp samples. This protocol enables the stabilization and preservation of nucleic acids in açaí, along with incorporating an exogenous internal amplification control. The standardization of the RNA extraction method involved a simple and reproducible approach, coupled with a one-step RT-qPCR assay. The assay underwent validation with various
DTUs and demonstrated sensitivity in detecting up to 0.1 viable parasite equivalents/mL in açaí samples. Furthermore, we investigated the effectiveness of a bleaching method in eliminating viable parasites in açaí samples contaminated with
by comparing the detection of DNA versus RNA. Finally, we validated this methodology using açaí pulp samples positive for
DNA, which were collected in a municipality with a history of oral CD outbreaks (Coari-AM). This validation involved comparing the detection and quantification of total versus viable
. Collectively, our findings demonstrate the feasibility of this methodology in detecting viable forms of
in açaí pulp samples, emerging as a crucial tool for monitoring oral outbreaks of Chagas disease resulting from açaí consumption.
In Brazil, orally acquired T. cruzi infection has become the most relevant transmission mechanisms from public health perspective. Around 70% of new Chagas disease cases have been associated with ...consumption of contaminated food or beverages. Açai (Euterpe oleracea and Euterpe precatoria) is currently one of the most commercialized Amazonian fruits in the Brazilian and international markets. Therefore, it has become important to incorporate in the production process some procedures to measure out effective hygiene and product quality control required by global market. Molecular methods have been developed for rapid detection and quantification of T. cruzi DNA in several biological samples, including food matrices, for epidemiological investigation of Chagas disease and food quality control. However, a high-performance molecular methodology since DNA extraction until detection and quantification of T. cruzi DNA in açai berry pulp is still needed. Herein, a simple DNA extraction methodology was standardized from the supernatant of açai berry pulp stabilized in a 6M Guanidine-HCl/0.2M EDTA buffer. In addition, a multiplex real time qPCR assay, targeting T. cruzi DNA and an Exogenous Internal Positive Control was developed and validated, using reference from all T. cruzi DTUs and commercial samples of açai pulp, from an endemic municipality with previous history of oral Chagas disease outbreak. Thus, a high-sensitivity qPCR assay, that could detect up to 0.01 parasite equivalents/mL in açai, was reached. As of the 45 commercial samples analyzed, 9 (20%) were positive for T. cruzi. This high-sensitive, fast, and easy-to-use molecular assay is compatible with most of the laboratories involved in the investigations of oral Chagas disease outbreaks, representing an important tool to the epidemiology, control, and surveillance of Chagas disease.
A recurring question concerning Trypanosoma cruzi DNA detection/quantification is related to the fact that DNA amplification, by itself, does not differentiate between viable or dead parasites. On ...the other hand, RNA can be considered a potential molecular marker of pathogens viability. Herein, we developed a quantitative real-time PCR with reverse Transcription (RT-qPCR) to quantify viable T. cruzi in artificially infected Rhodnius prolixus whilst evaluating differences between DNA and mRNA quantification along the insect midgut during 5, 9, 15 and 29 days after feeding. The RT-qPCR presented an improved performance with linearities ranging from 10.sup.7 to 10.sup.2 parasites equivalents and 3 to 0.0032 intestine unit equivalents, and efficiencies of 100.3% and 102.8% for both T. cruzi and triatomine targets, respectively. Comparing both RT-qPCR and qPCR, we confirmed that RNA is faster degraded, no longer being detected at day 1 after parasite lysis, while DNA detection was stable, with no decrease in parasite load over the days, even after parasite lysis. We also observed statistical differences between the quantification of the parasite load by DNA and by RNA on day 15 after feeding of experimentally infected R. prolixus. When assessing different portions of the digestive tract, by RT-qPCR, we could detect a statistically significant reduction in the parasite amount in the anterior midgut. Oppositely, there was a statistically significant increase of the parasite load in the hindgut. In conclusion, for this study parasite's viability in R. prolixus digestive tract were assessed targeting T. cruzi mRNA. In addition, differences between DNA and RNA detection observed herein, raise the possibility that RNA is a potential molecular viability marker, which could contribute to understanding the dynamics of the parasite infection in invertebrate hosts.
Several cases of food-borne acute Chagas disease (ACD) have been reported in the Brazilian Amazon so far. Up to 2004, the occurrence of ACD by oral transmission, associated with food consumption, was ...rare. Recent cases of ACD in Brazil have been attributed to the consumption of juice from the açai palm containing reservoir animals or insect vectors waste, infected with Trypanosoma cruzi. This study aimed to determine the T. cruzi contamination rate and to genotype the parasite in food samples prepared from açai, which are commercialized in Rio de Janeiro and the Pará States in Brazil.
The amplificability of DNA extracted from açai samples, and T. cruzi and Triatominae detection were performed by conventional PCR. Molecular characterization was done by multilocus PCR analysis, to determine the parasite discrete type units (DTUs) based on the size of PCR products in agarose gels, using the intergenic region of the spliced leader (SL), 24 Sα rDNA and nuclear fragment A10 as targets.
From the 140 samples of açai-based products analyzed, T. cruzi DNA was detected in 14 samples (10%); triatomine DNA was detected in one of these 14 samples. The parasite genotyping demonstrated that food samples containing açai showed a mixture of T. cruzi DTUs with TcIII, TcV and TcI prevailing.
In this study, the molecular detection and identification of T. cruzi from açai-based manufactured food samples, was performed for the first time. Although parasite DNA is a marker of possible contamination during food manufacturing, our findings do not indicate that açai is a source of Chagas disease via oral transmission per se, as live parasites were not investigated. Nevertheless, a molecular approach could be a powerful tool in the epidemiological investigation of outbreaks, supporting previous evidence that açai-based food can be contaminated with T. cruzi. Furthermore, both food quality control and assessment of good manufacturing practices involving açai-based products can be improved, assuring the safety of açai products.
is a sylvatic vector species in Brazil. In We aimed to characterize the
discrete typing units (DTUs), the parasitic loads, and the blood meal sources of insects collected in rocky outcrops in rural ...areas in the state of Minas Gerais. An optical microscope (OM) and kDNA-PCR were used to examine natural infection by
, and positive samples were genotyped by conventional multilocus PCR. Quantification of the
load was performed using qPCR, and the blood meal sources were identified by Sanger sequencing the 12S rRNA gene. A total of 141
were captured. Of these, ~55% (61/111) and ~91% (63/69) were positive by OM and KDNA-PCR, respectively. We genotyped ~89% (56/63) of the
-positive triatomines, with TcI (~55%, 31/56) being the most prevalent DTU, followed by TcIII (~20%, 11/56) and TcII (~7%, 4/56). Only TcI+TcIII mixed infections were detected in 10 (~18%) specimens. A wide range of variation in the parasitic loads of
was observed, with an overall median value of 10
parasites/intestine, with females having higher
loads than N2, N4, and N5. TcII showed lower parasitic loads compared to TcI and TcIII. The OM positive diagnosis odds ratio between
infection when the parasite load is 10
compared to 10
was approximately 29.1. The most frequent blood meal source was
(~58%), followed by
(~18%),
(~8%),
(~8%) and
(~8%). Our findings characterize biological and epidemiological aspects of the sylvatic population of
in the study area, highlighting the need to extend surveillance and control to this vector.
Chagas disease is a complex anthropozoonosis with distinct domestic and sylvatic mammal species acting as potential reservoirs. The diversity of vector species and their habitats are among the ...factors that hinder the control of the disease. Control programs periodically monitor the prevalence of T. cruzi infection in insect bugs through microscopical observation of diluted feces. However, microscopy presents limited sensitivity in samples with low parasite numbers, difficulties in examining all evolutionary stages of the insect and may in turn be limited to differentiate T. cruzi from other morphologically similar trypanosomatids. Here, we report two highly sensitive and accurate methodologies to infer T. cruzi infection rates and to quantify parasite load in the gut of field-collected triatomines.
Triatomines were manually collected in the period 2011-2012 and 2014-2015, in domestic, peridomestic or sylvatic habitats in rural areas of 26 municipalities, encompassing three distinct Brazilian biomes: Caatinga, Cerrado and Atlantic Rainforest. Following morphological and taxonomical identification, the search for flagellated protozoa was performed by optical microscopy. A conventional PCR targeting T. cruzi kDNA and a TaqMan qPCR directed to the parasite nuclear satellite DNA (SAT) were developed, both in multiplex, with the triatomine 12S subunit ribosomal RNA gene, used as internal amplification control. Both methods were used for detection (kDNA-PCR) and parasite load quantification (SAT-DNA-qPCR), to investigate T. cruzi infection in captured triatomines.
The combined methods were assayed on a panel of 205 field-collected triatomine samples. Diagnostic analysis revealed 21% positivity for the kDNA-PCR, whereas microscopic examination enabled identification of T. cruzi in only 7.0% of the PCR-positive samples. Negative PCR results were confirmed by the absence of T. cruzi flagellates using microscopy. Caatinga biome yielded the highest T. cruzi infection rate (60%), followed by the Atlantic Rainforest and Cerrado with 7.1 and 6.1%, respectively. In addition, a wide range distribution of parasite load, varying from 8.05 × 10
to 6.31 × 10
was observed with a median of 2.29 × 10
T. cruzi/intestine units. When parasite load was analyzed by triatomine species, a significantly higher median was found for Panstrongylus lutzi in comparison with Triatoma brasiliensis.
Our results demonstrate highly sensitive PCR-based methodologies to monitor T. cruzi infection in triatomines. In addition, the qPCR assay offers the possibility of further evaluation parasite load, as a promising biomarker of the vectorial capacity of triatomines in Chagas disease endemic areas.
Currently, approximately 70% of new cases of Chagas disease (CD) in Brazil are attributed to oral transmission, particularly through foods such as açaí, bacaba, and sugarcane juice, primarily in the ...northern and northeastern regions of the country. This underscores the imperative need to control the spread of the disease. The methods utilized to conduct quality control for food associated with outbreaks and to assess the potential for the oral transmission of CD through consuming açaí primarily rely on isolating the parasite or inoculating food into experimental animals, restricting the analyses to major research centers. While there are existing studies in the literature on the detection and quantification of T. cruzi DNA in açaí, the evaluation of parasites’ viability using molecular methods in this type of sample and differentiating between live and dead parasites in açaí pulp remain challenging. Consequently, we developed a molecular methodology based on RT-qPCR for detecting and quantifying viable T. cruzi in açaí pulp samples. This protocol enables the stabilization and preservation of nucleic acids in açaí, along with incorporating an exogenous internal amplification control. The standardization of the RNA extraction method involved a simple and reproducible approach, coupled with a one-step RT-qPCR assay. The assay underwent validation with various T. cruzi DTUs and demonstrated sensitivity in detecting up to 0.1 viable parasite equivalents/mL in açaí samples. Furthermore, we investigated the effectiveness of a bleaching method in eliminating viable parasites in açaí samples contaminated with T. cruzi by comparing the detection of DNA versus RNA. Finally, we validated this methodology using açaí pulp samples positive for T. cruzi DNA, which were collected in a municipality with a history of oral CD outbreaks (Coari-AM). This validation involved comparing the detection and quantification of total versus viable T. cruzi. Collectively, our findings demonstrate the feasibility of this methodology in detecting viable forms of T. cruzi in açaí pulp samples, emerging as a crucial tool for monitoring oral outbreaks of Chagas disease resulting from açaí consumption.
Levamisole (Lms) is an anthelminthic drug with immunomodulatory activity. Chagas disease (CD) is caused by Trypanosoma cruzi and there is very low access to the drugs available, benznidazole (Bz) and ...nifurtimox, both far from ideal. In a drug-repurposing strategy to test potential activity as antiparasitic and immunomodulatory agent for CD, Lms was assayed on acute T. cruzi murine infection, alone and in co-administration with Bz. During protocol standardization, 100 and 10 mpk of Bz given for five consecutive days resulted in parasitaemia suppression and 100% animal survival only with the highest dose. Flow cytometry showed that both optimal (100 mpk) and suboptimal (10 mpk) doses of Bz equally decreased the plasma levels of cytokines commonly elevated in this acute infection model. Lms alone (10-0.5 mpk) did not decrease parasitaemia nor mortality rates. Co-administration was investigated using the suboptimal dose of Bz and different doses of Lms. While Bz 10 mpk did not alter parasitaemia, the combo partially reduced it but only slightly promoted animal survival. This effect could be related to Th1-response modulation since interleukin-6 and interferon-γ were higher after treatment with the combo.
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