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

ABSTRACT: BACKGROUND: Chagas 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. RESULTS: The 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 IC(50) ranging from 10.49 to 93.7 ?M. 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. CONCLUSION: Here, we propose NTR as the major marker of the appearance of resistance to BZ.

SUBMITTER: Mejia-Jaramillo AM

PROVIDER: S-EPMC3185274 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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

Mejía-Jaramillo Ana M AM Fernández Geysson J GJ Palacio Lina L Triana-Chávez Omar O

Parasites & vectors 20110905

<h4>Background</h4>Chagas 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.<h4>Results</h4>The ...[more]

PMID: 21892937

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Project description:This study was performed to comparably assess two commercial real-time PCR assays for the identification of Trypanosoma cruzi DNA in serum. A total of 518 Colombian serum samples with high pre-test probability for infections with either T. cruzi or apathogenic Trypanosoma rangeli were assessed. The assessment comprised the NDO real-time PCR (TIB MOLBIOL, ref. no. 53-0755-96, referred to as the TibMolBiol assay in the following) with specificity for T. cruzi and the RealStar Chagas PCR Kit 1.0 (altona DIAGNOSTICS, order no. 611013, referred to as the RealStar assay in the following) targeting a kinetoplast sequence of both T. cruzi and T. rangeli without further discrimination. To discriminate between T. cruzi- and T. rangeli-specific real-time PCR amplicons, Sanger sequencing results were available for a minority of cases with discordant real-time PCR results, while the amplicons of the remaining discordant samples were subjected to nanopore sequencing. The study assessment indicated a proportion of 18.1% (n = 94) T. cruzi-positive samples next to 24 samples (4.6%) containing DNA of the phylogenetically related but apathogenic parasite T. rangeli. The observed diagnostic accuracy as expressed by sensitivity and specificity was 97.9% (92/94) and 99.3% (421/424) with the TibMolBiol assay and 96.8% (91/94) and 95.0% (403/424) with the RealStar assay, respectively. Reduced specificity resulted from cross-reaction with T. rangeli in all instances (3 cross-reactions with the TibMolBiol assay and 21 cross-reactions with the RealStar assay). DNA from the six discrete typing units (DTUs) of T. cruzi was successfully amplified by both real-time PCR assays. In summary, both assays showed a comparable diagnostic accuracy for the diagnosis of T. cruzi from human serum, with a slightly higher specificity seen for the TibMolBiol assay. The pronounced co-amplification of DNA from apathogenic T. rangeli according to the RealStar assay may be a disadvantage in areas of co-circulation with T. cruzi, while the test performance of the two compared assays will be quite similar in geographic settings where T. rangeli infections are unlikely.

Project description: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 × 10-2 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.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.

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

Publications

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

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