Towards a New Strategy for Diagnosis of Congenital Trypanosoma cruzi Infection.

The immigration of Latin American women of childbearing age has spread the congenital transmission of Chagas disease to areas of nonendemicity, and the disease is now a worldwide problem. Some European health authorities have implemented screening programs to prevent vertical transmission, but the lack of a uniform protocol calls for the urgent establishment of a new strategy common to all laboratories. Our aims were to (i) analyze the trend of passive IgG antibodies in the newborn by means of five serological tests for the diagnosis and follow-up of congenital Trypanosoma cruzi infection, (ii) assess the utility of these techniques for diagnosing a congenital transmission, and (iii) propose a strategy for a prompt, efficient, and cost-effective diagnosis of T. cruzi infection. In noninfected newborns, a continuous decreasing trend of passive IgG antibodies was observed, but none of the serological assays seroreverted in any the infants before 12 months. From 12 months onwards, serological tests achieved negative results in all the samples analyzed, with the exception of the highly sensitive chemiluminescent microparticle immunoassay (CMIA). In contrast, in congenitally infected infants, the antibody decline was detected only after treatment initiation. In order to improve the diagnosis of congenital T. cruzi infection, we propose a new strategy involving fewer tests that allows significant cost savings. The protocol could start 1 month after birth with a parasitological test and/or a PCR. If negative, a serological test would be carried out at 9 months, which if positive, would be followed by another at around 12 months for confirmation.

Serological Diagnosis of Chronic Chagas Disease: Is It Time for a Change?

Chagas disease has spread to areas that are nonendemic for the disease with human migration. Since no single reference standard test is available, serological diagnosis of chronic Chagas disease requires at least two tests. New-generation techniques have significantly improved the accuracy of Chagas disease diagnosis by the use of a large mixture of recombinant antigens with different detection systems, such as chemiluminescence. The aim of the present study was to assess the overall accuracy of a new-generation kit, the Architect Chagas (cutoff, ≥1 sample relative light units/cutoff value [S/CO]), as a single technique for the diagnosis of chronic Chagas disease. The Architect Chagas showed a sensitivity of 100% (95% confidence interval [CI], 99.5 to 100%) and a specificity of 97.6% (95% CI, 95.2 to 99.9%). Five out of six false-positive serum samples were a consequence of cross-reactivity with Leishmania spp., and all of them achieved results of <5 S/CO. We propose the Architect Chagas as a single technique for screening in blood banks and for routine diagnosis in clinical laboratories. Only gray-zone and positive sera with a result of ≤6 S/CO would need to be confirmed by a second serological assay, thus avoiding false-positive sera and the problem of cross-reactivity with Leishmania species. The application of this proposal would result in important savings in the cost of Chagas disease diagnosis and therefore in the management and control of the disease.

Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays.

BACKGROUND: Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. CONCLUSIONS/SIGNIFICANCE: This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.