Dados do Trabalho

Título

Analysis in vitro and in vivo of RNA as a molecular marker of Trypanosoma cruzi viability

Introdução

Molecular methods for T. cruzi detection or quantification present advantages over optical microscopy, the classical method of diagnosis in triatomine samples. Nevertheless, a recurring question concerning T. cruzi DNA detection/quantification is mainly because DNA amplification does not differentiate between viable or dead parasites. On the other hand, the RNA, due to its half-life and lability, can be considered a potential molecular marker of pathogens viability.

Objetivo (s)

Until now there are few reports comparing the application of molecular diagnostic tools that effectively differentiate between viable and nonviable parasites especially when it comes to T. cruzi evaluation. Herein, we aimed to evaluate RNA as a marker of T. cruzi viability both in vivo, with Rhodnius prolixus, and in vitro.

Material e Métodos

Therefore, we developed a qPCR with reverse Transcription (RT-qPCR), targeting T. cruzi GAPDH mRNA, a gene constitutively expressed among T. cruzi developmental stages and DTUs, to quantify viable parasites while comparing with the qPCR, targeting T. cruzi satellite DNA.

Resultados e Conclusão

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. Regarding R. prolixus experimentally infected, we observed a decrease in parasite load through mRNA detection on days 5, 9 15 and 29 after feeding, which is not followed by DNA quantification, especially on day 15 after feeding. Moreover, when assessing different portions of the digestive tract, by RT-qPCR, we could detect a statistically significant reduction of the parasite amount in the anterior midgut on day 29 after feeding. Oppositely, we could observe a statistically significant increase of parasite load in the hindgut on the same day. Comparing both methods in in vitro infection assays, we confirmed that RNA is shortly degraded after parasite lysis, while parasite DNA detection presented no significant decrease over the days, even after parasite lysis. In conlusion, differences between DNA and RNA detection observed herein, raises the possibility that RNA is a potential molecular viability marker, which could contribute to understanding the dynamics of the parasite infection in vertebrate and invertebrate hosts.

Palavras-chave

Trypanosoma cruzi; viability; molecular diagnosis, RT-qPCR, qPCR

Agradecimentos

FIOCRUZ; FAPERJ; CNPq