02500nas a2200337   4500000000100000008004100001653003900042653001900081653002900100653002000129653001400149100001200163700001300175700001200188700001700200700001600217700001100233700002200244700001400266700001300280700001300293700001100306700001400317700004700331245008500378856009900463300001300562490000700575520156600582022001402148       2019                            d10aNeglected tropical diseases (NTDs)10aChagas disease10aDNA extraction technique10aDiagnostic tool10adiagnosis1 aMayta H1 aRomero Y1 aPando A1 aVerastegui M1 aTinajeros F1 aBozo R1 aHenderson-Frost J1 aColanzi R1 aFlores J1 aLerner R1 aBern C1 aGilman RH1 aChagas Working Group in Perú and Bolivia 00aImproved DNA extraction technique from clot for the diagnosis of Chagas disease.  uhttps://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0007024&type=printable  ae00070240 v133 a<p><strong>BACKGROUND: </strong>The 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.</p>

<p><strong>METHODOLOGY/PRINCIPAL FINDINGS: </strong>A 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.</p>

<p><strong>CONCLUSIONS: </strong>The new methodology for DNA extraction from clot samples improves the molecular diagnosis of Chagas disease.</p>
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