Sensitive and Specific Detection of Trypanosoma cruzi DNA in Clinical Specimens Using a Multi-Target Real-Time PCR Approach

Abstract

Background The laboratory diagnosis of Chagas disease is challenging because the usefulness of different diagnostic tests will depend on the stage of the disease. Serology is the preferred method for patients in the chronic phase, whereas PCR can be successfully used to diagnose acute and congenital cases. Here we present data using a combination of three TaqMan PCR assays to detect T. cruzi DNA in clinical specimens. Methods/Principal Findings Included in the analysis were DNA extracted from 320 EDTA blood specimens, 18 heart tissue specimens, 6 umbilical cord blood specimens, 2 skin tissue specimens and 3 CSF specimens. For the blood specimens both whole blood and buffy coat fraction were analyzed. The specimens were from patients living in the USA, with suspected exposure to T. cruzi through organ transplantation, contact with triatomine bugs or laboratory accidents, and from immunosuppressed patients with suspected Chagas disease reactivation. Real-time PCR was successfully used to diagnose acute and Chagas disease reactivation in 20 patients, including one case of organ-transmitted infection and one congenital case. Analysis of buffy coat fractions of EDTA blood led to faster diagnosis in six of these patients compared to whole blood analysis. The three real-time PCR assays produced identical results for 94% of the specimens. The major reason for discrepant results was variable sensitivity among the assays, but two of the real-time PCR assays also produced four false positive results. Conclusions/Significance These data strongly indicate that at least two PCR assays with different performances should be combined to increase the accuracy. This evaluation also highlights the benefit of extracting DNA from the blood specimen's buffy coat to increase the sensitivity of PCR analysis.

Inhibitory Receptor Expression on CD8+ T Cells Is Linked to Functional Responses against Trypanosoma cruzi Antigens in Chronic Chagasic Patients

“…Although the parasite burden was nonquantifiable in this study, parasite DNA was found in the peripheral blood of all groups of chagasic patients, indicating parasite persistence. During chronic infection, the parasite remains in tissues, such as the heart and other reservoir tissues (94)(95)(96)(97); thus, the parasite load in the blood is low or not detectable in some patients. Indeed, the antigenic load is an important difference between viral and parasitic models of CD8 + T cell dysfunction.…”

Characterization and Diagnostic Application of Trypanosoma cruzi Trypomastigote Excreted-Secreted Antigens Shed in Extracellular Vesicles Released from Infected Mammalian Cells

“…Microscopy and PCR-based methods are more effective for diagnosing acute or congenital forms of Chagas disease (14,15), while serological tests using either parasite-derived antigens, recombinant proteins, or synthetic peptides are preferred for diagnosis of chronic infections (16). Despite the sensitivity of serological tests, current Chagas disease diagnostic tests may lack specificity due to cross-reactivity with the related parasites Leishmania spp.…”

Evaluation of Nifurtimox Treatment of Chronic Chagas Disease by Means of Several Parasitological Methods

“…However, it must be underlined that the validity of molecular and parasitological methods relies on the positive results they can give, and accordingly they have been proposed for earlier assessment of treatment failure of treated chronic Chagas disease patients (15). T. cruzi contains nuclear DNA and kinetoplast DNA (kDNA), both of which contain many repetitive sequences that are highly suitable for sensitive PCR detection due to their high copy numbers (16,17). Other parasitological methods, such as the classical XD method (18), even though of much less sensitivity than PCR, are still useful in combination with PCR (19,20).…”