Detection of Trypanosoma cruzi in untreated chronic chagasic patients is improved by using three parasitological methods simultaneously

Zulantay, Inés; Apt, Werner; Valencia, Claudio; Torres, A; Saavedra, Miguel; Rodríguez, Jorge; Sandoval, L; Martínez, Gabriela; Thieme, Patricio; Sepúlveda, Eduardo

doi:10.1093/jac/dkr313

Cited by 15 publications

(14 citation statements)

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“…The application of the polymerase chain reaction (PCR) has allowed the detection of Trypanosoma cruzi in several biological samples obtained from insect vectors and mammalian hosts [1-5]; however, it is a qualitative technique. By contrast, the real-time PCR (qPCR) technique allows detection and quantification of T. cruzi parasitic loads, which is particularly important in studies where the effectiveness of chemotherapy is being evaluated [6,7].…”

Section: Resultsmentioning

confidence: 99%

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Real-Time PCR in faecal samples of Triatoma infestans obtained by xenodiagnosis: proposal for an exogenous internal control

Bravo

Muñoz

Nazal³

et al. 2012

Parasites Vectors

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BackgroundThe polymerase chain reaction (PCR) has proved to be a sensitive technique to detect Trypanosoma cruzi in the chronic phase of Chagas disease, which is characterized by low and fluctuating parasitemia. Another technique proposed for parasitological diagnosis in this phase of infection combines a microscopic search for motile trypomastigote forms in faecal samples (FS) obtained by xenodiagnosis (XD) with conventional PCR (XD-PCR). In this study we evaluate the use of human blood DNA as an exogenous internal control (EIC) for real time PCR (qPCR) combined with XD (XD-qPCR) using chromosome 12 (X12) detection.FindingsNone of the FS-XD evaluated by qPCR amplified for X12. Nevertheless, all the EIC-FS-XD mixtures amplified for X12.ConclusionsWe determined that X12 is useful as an EIC for XD-qPCR because we showed that the FS-XD does not contain human DNA after 30 or more days of XD incubation. This information is relevant for research on T. cruzi by XD-qPCR since it allows ruling out inhibition and false negative results due to DNA loss during the process of extraction and purification.

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Section: Resultsmentioning

confidence: 99%

“…The FS-XD were received in 500 μl PBS buffer pH 7.2, incubated for 15 min at 98°C in a double boiler and centrifuged for 3 min at 3500 rpm. A total of 200 μl of supernatant from each FS-XD was collected and stored at -20°C until use [5]. …”

Section: Resultsmentioning

confidence: 99%

Real-Time PCR in faecal samples of Triatoma infestans obtained by xenodiagnosis: proposal for an exogenous internal control

Bravo

Muñoz

Nazal³

et al. 2012

Parasites Vectors

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“…Acute CD phase, which lasts for ~ ten weeks, is relatively easy to diagnose by microscopy, polymerase chain reaction (PCR), hemoculture or/and xenodiagnoses [14]. However, the vast majority of CD cases are not recognized and the disease enters the chronic phase when these tests targeting parasite material in the blood become unreliable due to the low and variable levels of parasitemia.…”

Section: Introductionmentioning

confidence: 99%

Serum biomarkers predictive of cure in Chagas disease patients after nifurtimox treatment

et al. 2014

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BackgroundChagas disease (CD), caused by the protozoan Trypanosoma cruzi, remains an important public health issue in many Central and South American countries, as well as non-endemic areas with high rates of immigration from these countries. Existing treatment options for CD are limited and often unsatisfactory. Moreover the lack of post-treatment tests of cure limits the development of new drugs. To address this issue, we sought to identify serum biomarkers following nifurtimox (Nfx) treatment that could be used as an early test of cure and/or markers of a therapeutic response.MethodsHuman sera from Chagas patients pre- and post-treatment with Nfx (n = 37) were compared to samples from healthy subjects (n = 37) using a range of proteomic and immunologic techniques. Biomarker peaks with the best discriminatory power were further characterized.ResultsUsing serum samples (n = 111), we validated the presence of five key biomarkers identified in our previous study, namely human apolipoprotein A-I (APOA1) and specific fragments thereof and one fragment of human fibronectin (FN1). In chagasic serum samples all biomarkers except full-length APOA1 were upregulated. These five biomarkers returned to normal in 43% (16/37) of the patients treated with Nfx at three years after treatment.ConclusionsThe normalization of biomarker patterns strongly associated with CD suggests that these markers can be used to identify patients in whom Nfx treatment is successful. We believe that these are the first biomarkers predictive of cure in CD patients.

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“…Thus, recently infected individuals may obtain a false negative result by PCR. Similarly, during the chronic phase of disease the number of circulating parasites in blood can fluctuate over time and be difficult to detect by PCR [6], [7]. Under these conditions, if a single parasite is not present in the volume of blood sample used for testing, a negative PCR result will be obtained.…”

Section: Introductionmentioning

confidence: 99%

Development of an Aptamer-Based Concentration Method for the Detection of Trypanosoma cruzi in Blood

et al. 2012

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Trypanosoma cruzi, a blood-borne parasite, is the etiological agent of Chagas disease. T. cruzi trypomastigotes, the infectious life cycle stage, can be detected in blood of infected individuals using PCR-based methods. However, soon after a natural infection, or during the chronic phase of Chagas disease, the number of parasites in blood may be very low and thus difficult to detect by PCR. To facilitate PCR-based detection methods, a parasite concentration approach was explored. A whole cell SELEX strategy was utilized to develop serum stable RNA aptamers that bind to live T. cruzi trypomastigotes. These aptamers bound to the parasite with high affinities (8–25 nM range). The highest affinity aptamer, Apt68, also demonstrated high specificity as it did not interact with the insect stage epimastigotes of T. cruzi nor with other related trypanosomatid parasites, L. donovani and T. brucei, suggesting that the target of Apt68 was expressed only on T. cruzi trypomastigotes. Biotinylated Apt68, immobilized on a solid phase, was able to capture live parasites. These captured parasites were visible microscopically, as large motile aggregates, formed when the aptamer coated paramagnetic beads bound to the surface of the trypomastigotes. Additionally, Apt68 was also able to capture and aggregate trypomastigotes from several isolates of the two major genotypes of the parasite. Using a magnet, these parasite-bead aggregates could be purified from parasite-spiked whole blood samples, even at concentrations as low as 5 parasites in 15 ml of whole blood, as detected by a real-time PCR assay. Our results show that aptamers can be used as pathogen specific ligands to capture and facilitate PCR-based detection of T. cruzi in blood.

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Detection of Trypanosoma cruzi in untreated chronic chagasic patients is improved by using three parasitological methods simultaneously

Abstract: The simultaneous application of more than one technique for the parasitological diagnosis of Chagas' disease in untreated individuals increases the possibility of detection of T. cruzi.

Cited by 15 publications

References 10 publications

Real-Time PCR in faecal samples of Triatoma infestans obtained by xenodiagnosis: proposal for an exogenous internal control

Real-Time PCR in faecal samples of Triatoma infestans obtained by xenodiagnosis: proposal for an exogenous internal control

Serum biomarkers predictive of cure in Chagas disease patients after nifurtimox treatment

Development of an Aptamer-Based Concentration Method for the Detection of Trypanosoma cruzi in Blood

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