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Investigation of molecular diagnostic assays for the detection of Trypanosoma cruzi DNA in blood

Abstract number: O42

Alvarez-Martinez M.J., Nolder D., Chiodini P.

Objectives: Chagas disease, caused by Trypanosoma cruzi, is endemic to Latin America, and of emerging importance in non-endemic countries because of migration of people infected with T. cruzi. The majority of patients diagnosed in non-endemic settings are in the indeterminate or chronic phases. However, acute cases may be seen in congenitally-infected infants and in people receiving blood products or organs from infected donors. Molecular diagnostic assays needed to be standardized to diagnose and monitor congenital infection, aid serological diagnosis in expatriate travellers and migrants from Latin America, and monitor infection in known cases of Chagas Disease.

Methods: Two molecular assays for the diagnosis of T. cruzi infection were investigated, a SYBR green real-time PCR targeting nuclear satellite DNA, and a hotstart PCR targeting kinetoplast DNA (kDNA). One hundred and eighty PCR controls and 180 blood-simulated specimens prepared from cultures of T. cruzi strains of lineages TcI, TcIIb and TcIIe were evaluated. Samples for DNA were prepared from the specimens using 3 lysis methods (Qiagen (Q), Guanidine-EDTA (GE) and GE+boiling (GEB). DNA extraction after lysis was carried out using silica-membrane technology. Ten-fold serial dilutions were prepared. Statistical analysis was performed by SPSS v15, Chicago IL.

Results: The sensitivity of the real-time PCR assay was calculated by PROBIT analysis with 12 replicates of 8-fold serial dilutions of the DNA from culture of TcIIb extracted by Qiagen. The 95% and 50% positive hit rates were 0.8 parasites/ml (95% IC: 0.41–1.53) and 0.2 parasites/ ml (95% IC: 0.08–0.32), respectively. The assay showed a specificity of 100%. More consistent results were found for all lineages and types of samples when DNA was extracted by the Qiagen method. A higher detection limit was found in lineage TcI (p < 0.01). Lineage TcIIb showed statistically better results in controls and simulated specimens, than the other two lineages (p < 0.01). The kDNA assay was performed in simulated specimens from the three lineages, and it gave better results using Q and GEB methods on low concentration samples, although not statistically significant (p = 0.08).

Conclusions: Molecular assays are very promising in the diagnosis of T. cruzi infection and have applications where serological assays are not of use. Further work should be done to standardize the methods.

Session Details

Date: 10/04/2010
Time: 00:00-00:00
Session name: Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Vienna, Austria, 10 - 13 April 2010
Presentation type:
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