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.

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.

Nitric oxide effects on Rhodnius prolixus's immune responses, gut microbiota and Trypanosoma cruzi development.

The immune system of Rhodnius prolixus comprehends the synthesis of different effectors that modulate the intestinal microbiota population and the life cycle of the parasite Trypanosoma cruzi inside the vector midgut. One of these immune responses is the production of reactive nitrogen species (RNS) derived by the action of nitric oxide synthase (NOS). Therefore, we investigated the effects of L-arginine, the substrate for nitric oxide (NO) production and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NOS, added in the insect blood meal. We analyzed the impact of these treatments on the immune responses and development of intestinal bacteria and parasites on R. prolixus nymphs. The L-arginine treatment in R. prolixus nymphs induced a higher NOS gene expression in the fat body and increased NO production, but reduced catalase and antimicrobial activities in the midgut. As expected, L-NAME treatment reduced NOS gene expression in the fat body. In addition, L-NAME treatment diminished catalase activity in the hemolymph and posterior midgut reduced phenoloxidase activity in the anterior midgut and increased the antimicrobial activity in the hemolymph. Both treatments caused a reduction in the cultivatable intestinal microbiota, especially in insects treated with L-NAME. However, T. cruzi development in the insect's digestive tract was suppressed after L-arginine treatment and the opposite was observed with L-NAME, which resulted in higher parasite counts. Therefore, we conclude that induction and inhibition of NOS and NO production are associated with other R. prolixus humoral immune responses, such as catalase, phenoloxidase, and antibacterial activities in different insect organs. These alterations reflect on intestinal microbiota and T. cruzi development.

Everybody loves sugar: first report of plant feeding in triatomines.

BACKGROUND: Triatomines, which are the vectors of Trypanosoma cruzi, have been considered to be exclusive blood feeders for more than 100 years, since the discovery of Chagas disease. METHODS: We offered artificial sugar meals to the laboratory model-insect Rhodnius prolixus, which is considered a strict haematophagous insect. We registered feeding by adding colorant to sugar meals. To assess putative phytophagy, fruits of the tomato Solanum lycopersicum were offered to R. prolixus and the presence of tomato DNA was assessed in the insects using PCR. We also assessed longevity, blood feeding and urine production of fruit-exposed triatomines and control insects. RESULTS: All instars of R. prolixus ingested sugar from artificial sugar meals in laboratory conditions. First instar R. prolixus ingested plant tissue from S. lycopersicum fruits, and this increased the amount of blood ingested and urine excreted. Decreased mortality was also observed after blood feeding. Exposure to S. lycopersicum increased longevity and reduced weight loss caused by desiccation. CONCLUSIONS: We describe here the first report of sugar feeding and phytophagy in a species that was considered to be a strict blood-feeder for over a century. We suggest that local plants might be not merely shelters for insects and vertebrate hosts as previously described, but may have a nutritional role for the maintenance of the triatomine vectors. The description of sugar and plant meals in triatomines opens new perspectives for the study and control of Chagas Disease.

Characterization of the microbiota in the guts of Triatoma brasiliensis and Triatoma pseudomaculata infected by Trypanosoma cruzi in natural conditions using culture independent methods.

BACKGROUND: Chagas disease is caused by Trypanosoma cruzi, which is transmitted by triatomine vectors. The northeastern region of Brazil is endemic for Chagas disease and has the largest diversity of triatomine species. T. cruzi development in its triatomine vector depends on diverse factors, including the composition of bacterial gut microbiota. METHODS: We characterized the triatomines captured in the municipality of Russas (Ceará) by sequencing the cytochrome c oxidase subunit I (COI) gene. The composition of the bacterial community in the gut of peridomestic Triatoma brasiliensis and Triatoma pseudomaculata was investigated using culture independent methods based on the amplification of the 16S rRNA gene by polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), DNA fragment cloning, Sanger sequencing and 454 pyrosequencing. Additionally, we identified TcI and TcII types of T. cruzi by sequencing amplicons from the gut metagenomic DNA with primers for the mini-exon gene. RESULTS: Triatomines collected in the peridomestic ecotopes were diagnosed as T. pseudomaculata and T. brasiliensis by comparing their COI sequence with GenBank. The rate of infection by T. cruzi in adult triatomines reached 80% for T. pseudomaculata and 90% for T. brasiliensis. According to the DNA sequences from the DGGE bands, the triatomine gut microbiota was primarily composed of Proteobacteria and Actinobacteria. However, Firmicutes and Bacteroidetes were also detected, although in much lower proportions. Serratia was the main genus, as it was encountered in all samples analyzed by DGGE and 454 pyrosequencing. Members of Corynebacterinae, a suborder of the Actinomycetales, formed the next most important group. The cloning and sequencing of full-length 16S rRNA genes confirmed the presence of Serratia marcescens, Dietzia sp., Gordonia terrae, Corynebacterium stationis and Corynebacterium glutamicum. CONCLUSIONS: The study of the bacterial microbiota in the triatomine gut has gained increased attention because of the possible role it may play in the epidemiology of Chagas disease by competing with T. cruzi. Culture independent methods have shown that the bacterial composition of the microbiota in the guts of peridomestic triatomines is made up by only few bacterial species.