The Development of a One-Step RT-qPCR for the Detection and Quantification of Viable Forms of Trypanosoma cruzi in Açai Samples from Areas at Risk of Chagas Disease through Oral Transmission.

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.

Phenotypic Evaluation of Nucleoside Analogues against Trypanosoma cruzi Infection: In Vitro and In Vivo Approaches.

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.

Impact of levamisole in co-administration with benznidazole on experimental Chagas disease.

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.

Detection and Genotyping of Trypanosoma cruzi Samples in Species of Genus Rhodnius from Different Environments in the Brazilian Amazon.

Background: In the Amazon region, several species of triatomines occur in the natural environments. Among them, species of the genus Rhodnius are a risk to human populations due to their high rates of infection with Trypanosoma cruzi. The aim of this study was to identify the T. cruzi genotypes in Rhodnius specimens and their relationship with sylvatic hosts from different environments in the Brazilian Amazon. Methods: 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 T. cruzi in six discrete typing units (DTUs) was performed using conventional multilocus PCR. The triatomines that were positive for T. cruzi and engorged with blood were also targeted for amplification of the cytochrome B (cytB) gene to identify bloodmeal sources. Results: 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 Plecturocebus brunneus. TcIV was detected in triatomines that fed on Coendou prehensilis, Didelphis marsupialis, Mabuya nigropunctata, P. brunneus, Pithecia irrorata, Sapajus apella, and Tamandua tetradactyla. Conclusion: Results highlight the need to understand the patterns of T. cruzi genotypes in Rhodnius spp. and their association with sylvatic hosts to better elucidate their role in the transmission of Chagas disease in the Amazon region.

The Low Variability of Tc24 in Trypanosoma cruzi TcI as an Advantage for Chagas Disease Prophylaxis and Diagnosis in Mexico.

(1) Background: Chagas disease is the main neglected tropical disease in America. It is estimated that around 6 million people are currently infected with the parasite in Latin America, and 25 million live in endemic areas with active transmission. The disease causes an estimated economic loss of USD 24 billion dollars annually, with a loss of 75,200 working years per year of life; it is responsible for around ~12,000 deaths annually. Although Mexico is an endemic country that recorded 10,186 new cases of Chagas disease during the period of 1990-2017, few studies have evaluated the genetic diversity of genes that could be involved in the prophylaxis and/or diagnosis of the parasite. One of the possible candidates proposed as a vaccine target is the 24 kDa trypomastigote excretory-secretory protein, Tc24, whose protection is linked to the stimulation of T. cruzi-specific CD8+ immune responses. (2) Methods: The aim of the present study was to evaluate the fine-scale genetic diversity and structure of Tc24 in T. cruzi isolates from Mexico, and to compare them with other populations reported in the Americas with the aim to reconsider the potential role of Tc24 as a key candidate for the prophylaxis and improvement of the diagnosis of Chagas disease in Mexico. (3) Results: Of the 25 Mexican isolates analysed, 48% (12) were recovered from humans and 24% (6) recovered from Triatoma barberi and Triatoma dimidiata. Phylogenetic inferences revealed a polytomy in the T. cruzi clade with two defined subgroups, one formed by all sequences of the DTU I and the other formed by DTU II-VI; both subgroups had high branch support. Genetic population analysis detected a single (monomorphic) haplotype of TcI throughout the entire distribution across both Mexico and South America. This information was supported by Nei's pairwise distances, where the sequences of TcI showed no genetic differences. (4) Conclusions: Given that both previous studies and the findings of the present work confirmed that TcI is the only genotype detected from human isolates obtained from various states of Mexico, and that there is no significant genetic variability in any of them, it is possible to propose the development of in silico strategies for the production of antigens that optimise the diagnosis of Chagas disease, such as quantitative ELISA methods that use this region of Tc24.

RNA as a feasible marker of Trypanosoma cruzi viability during the parasite interaction with the triatomine vector Rhodnius prolixus (Hemiptera, Triatominae).

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 107 to 102 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.

High Parasitic Loads Quantified in Sylvatic Triatoma melanica, a Chagas Disease Vector.

Triatoma melanica is a sylvatic vector species in Brazil. In We aimed to characterize the Trypanosoma cruzi 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 T. cruzi, and positive samples were genotyped by conventional multilocus PCR. Quantification of the T. cruzi load was performed using qPCR, and the blood meal sources were identified by Sanger sequencing the 12S rRNA gene. A total of 141 T. melanica 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 T. cruzi-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 T. melanica was observed, with an overall median value of 104 parasites/intestine, with females having higher T. cruzi loads than N2, N4, and N5. TcII showed lower parasitic loads compared to TcI and TcIII. The OM positive diagnosis odds ratio between T. cruzi infection when the parasite load is 107 compared to 103 was approximately 29.1. The most frequent blood meal source was Kerodon rupestris (~58%), followed by Thrichomys apereoides (~18%), Wiedomys cerradensis (~8%), Galactis cuja (~8%) and Gallus gallus (~8%). Our findings characterize biological and epidemiological aspects of the sylvatic population of T. melanica in the study area, highlighting the need to extend surveillance and control to this vector.

Validation of a novel multiplex real-time PCR assay for Trypanosoma cruzi detection and quantification in açai pulp.

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.

Genotypic Trypanosoma cruzi distribution and parasite load differ ecotypically and according to parasite genotypes in Triatoma brasiliensis from endemic and outbreak areas in Northeastern Brazil.

This study aimed to identify the Trypanosoma cruzi genotypes and their relationship with parasitic load in distinct geographic and ecotypic populations of Triatoma brasiliensis in two sites, including one where a Chagas disease (ChD) outbreak occurred in Rio Grande do Norte state, Brazil. Triatomine captures were performed in peridomestic and sylvatic ecotopes in two municipalities: Marcelino Vieira - affected by the outbreak; and Currais Novos - where high pressure of peridomestic triatomine infestation after insecticide spraying have been reported. The kDNA-PCR was used to select 124 T. cruzi positive triatomine samples, of which 117 were successfully genotyped by fluorescent fragment length barcoding (FFLB). Moreover, the T. cruzi load quantification was performed using a multiplex TaqMan qPCR. Our findings showed a clear ecotypic segregation between TcI and TcII harboured by T. brasiliensis (p<0.001). Although no genotypes were ecotypically exclusive, TcI was predominant in peridomestic ecotopes (86%). In general, T. brasiliensis from Rio Grande do Norte had a higher T. cruzi load varying from 3.94 to 7.66 x 106T. cruzi per insect. Additionally, TcII (median value=299,504 T. cruzi/intestine unit equivalents) had more than twice (p=0.1) the parasite load of TcI (median value=149,077 T. cruzi/intestine unit equivalents), which can be attributed to a more ancient co-evolution with T. brasiliensis. The higher prevalence of TcII in the sylvatic T. brasiliensis (70%) could be associated with a more diversified source of bloodmeals for wild insect populations. Either TcI or TcII may have been responsible for the ChD outbreak that occurred in the city of Marcelino Vieira. On the other hand, a smaller portion of T. brasiliensis was infected by TcIII (3%) in the peridomicile, in addition to T. rangeli genotype A (1%), often found in mixed infections. Our results highlight the need of understanding the patterns of T. cruzi genotype´s development and circulation in insect vectors and reservoirs as a mode of tracking situations of epidemiologic importance, as the ChD outbreak recently recorded for Northeastern Brazil.

Development of conventional and real-time multiplex PCR-based assays for estimation of natural infection rates and Trypanosoma cruzi load in triatomine vectors.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 × 10-2 to 6.31 × 1010 was observed with a median of 2.29 × 103 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. CONCLUSIONS: 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.