Abstract
BackgroundThe detection of Trypanosoma cruzi genetic material in clinical samples is considered an important diagnostic tool for Chagas disease. We have previously demonstrated that PCR using clot samples yields greater sensitivity than either buffy coat or whole blood samples. However, phenol-chloroform DNA extraction from clot samples is difficult and toxic. The objective of the present study was to improve and develop a more sensitive method to recover parasite DNA from clot samples for the diagnosis of Chagas disease.Methodology/Principal findingsA total of 265 match pair samples of whole blood–guanidine (GEB) and clot samples were analyzed; 150 were from Chagas seropositive subjects. DNA was extracted from both whole blood-guanidine samples, using a previously standardized methodology, and from clot samples, using a newly developed methodology based on a combination of the FastPrep technique and the standard method for GEB extraction. A qPCR targeting the nuclear satellite sequences was used to compare the sample source and the extraction method. Of the 150 samples from Chagas positive individuals by serology, 47 samples tested positive by qPCR with DNA extracted by both GEB and clot, but an additional 13 samples tested positive only in DNA extracted from clot. No serology-negative samples resulted positive when tested by qPCR.ConclusionsThe new methodology for DNA extraction from clot samples improves the molecular diagnosis of Chagas disease.
Highlights
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is endemic in many parts of the Americas [1,2], where 6 to 7 million people are infected [3]
The new methodology for DNA extraction from clot samples improves the molecular diagnosis of Chagas disease
No differences on the Cq values were observed when quantitative PCR (qPCR) for T. cruzi was performed on DNA extracted using lysing matrix C, H or J from clot samples spiked with 5 x 106 parasites
Summary
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is endemic in many parts of the Americas [1,2], where 6 to 7 million people are infected [3]. Infected individuals enter a chronic phase where parasites are rarely seen in the blood and diagnosis relies on the use of serological tests. About 20–30% of infected individuals will develop cardiomyopathy during the subsequent chronic phase, the most important consequence of Chagas disease [1,2,5]. The test is highly sensitive for diagnosis of acute and congenital Chagas disease [1]. Systematic monitoring by means of PCR of serial blood specimens has been suggested to improve PCR diagnosis [1,7,8]. The detection of Trypanosoma cruzi genetic material in clinical samples is considered an important diagnostic tool for Chagas disease. The objective of the present study was to improve and develop a more sensitive method to recover parasite DNA from clot samples for the diagnosis of Chagas disease
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