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.Fil: Abras, Alba. Universidad de Barcelona; España. Universidad de Girona; España. Instituto de Salud Global de Barcelona; EspañaFil: Ballart, Cristina. Universidad de Barcelona; España. Instituto de Salud Global de Barcelona; EspañaFil: Llovet, Teresa. Universitat Autònoma de Barcelona; España. Hospital de la Santa Creu I Sant Pau; EspañaFil: Roig, Carme. Hospital de la Santa Creu I Sant Pau; EspañaFil: Gutiérrez, Cristina. Hospital de la Santa Creu I Sant Pau; EspañaFil: Tebar, Silvia. Universidad de Barcelona; España. Instituto de Salud Global de Barcelona; EspañaFil: Berenguer, Pere. Hospital de la Santa Creu I Sant Pau; EspañaFil: Pinazo, María-Jesús. Instituto de Salud Global de Barcelona; EspañaFil: Posada, Elizabeth. Instituto de Salud Global de Barcelona; EspañaFil: Gascón, Joaquim. Instituto de Salud Global de Barcelona; EspañaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gállego, Montserrat. Instituto de Salud Global de Barcelona; España. Universidad de Barcelona; EspañaFil: Muñoz, Carmen. Hospital de la Santa Creu I Sant Pau; España. Universitat Autònoma de Barcelona; Españ

Serological diagnosis of chronic Chagas disease: Is it time for a change?

Chagas disease has spread to non-endemic areas 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, Architect Chagas (cut-off ≥ 1 S/CO, sample relative light units/cut-off value), as a single technique in the diagnosis of chronic Chagas disease. Architect Chagas showed a sensitivity of 100% (95% confidence interval, CI = 99.5-100) and a specificity of 97.6% (95% CI = 95.2-99.9). Five out of six false-positive sera were a consequence of cross-reactivity with Leishmania spp. and all of them achieved results < 5 S/CO. We propose Architect Chagas as a single technique for screening in blood banks and for routine diagnosis in clinical laboratories. Only grey zone and positive sera with a result ≤ 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 spp. 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

Ácaros parásitos de micromamíferos en Cataluña. I. Familia Glycyphagidae

The study of 20 small mammals species -Rodentia. lnsectivora- collected in the Catalonian region, has allowed the discovery of 8 mite species of the family Glycyphagidae: Glycyphagus (Myacarus) hypudaei, Labidophorus talpae. Orycteroxenus dispar, O. soricis. Lophioglyphus liciosus, Sciuropsis eliomys, Xenoryctes punctatus and X. krameri, being S. eliomys a new species for Spain. Host relationships and distribution are discussed.</p

Ácaros parásitos de micromamíferos en Cataluña. II. Familia Listrophoridae

The analysis of 1.395 specimens of small mammals belonging to 20 species has allowed the dis­ covery of 5 acarine species of the family Listrophoridae: Afrolistrophorus apodemi, Listrophorus /euc­ karti, L. meditarreneus, L. meridiana/is and L. occitanus. This study has shown that the Listrophoridae have a strong endemic character and a great capacity to invade other hosts when the habitual of the species is present ·in the same biotop</p

Ácaros parásitos de micromamíferos en Cataluña. III. Familia Myocoptidae

The analysis of 20 small mammals species has allowed the discovery of 9 mite species belonging to the Myocoptidae family: Criniscansor apodemi, Myocoptes j. japonensis, M. musculinus, M. squamosus, Trichoecius apodemi, T. c/ethrionomydis, T. pitymydis, T. romboutsi and T. tenax. This study has shown that the Myocoptidae have a low frequency, a high degree of specificity, and a pronounced focalization in the case of C. apodemi and T. apodemi.</p

Primera denuncia de flebotomos (Diptera, Psychodidae, Phlebotominae) en la provincia de Lérida (España, Cataluña)

The prospection taken in Lerida province (Catalonia, Spain), with the adhesive papers method, has allowed, for the first time in this place, the discovery of flebotomine. In a papers area of 89,2 m2 , 7796 specimens belonging to 5 species were capturad: Phlebotomus papatasi ( 128), P. sergenti (217), P. ariasi (1357), P. perniciosus (1979) and Sergentomyia minuta (4115).</p

Enzymatic heterogeneity among strains of <i>Leishmania infantum</i> from human visceral and cutaneous leishmaniosis in Catalonia

The presence of Sarcocystis spp. was surveyed in 97 sheep in the province of Cordoba. Three methods were used for detection of the parasite in three different muscular location (heart, esophagus and diaphragmatic muscle). Sarcocystis bradyzoites were found in 85,6% of the sheep by trypsin digest. Microscopis S. tenella in all infected animals and S. arieticanis int he diaphragmatic muscle of one sheep, and macroscopic S. gigantea in the esophagus of 6 sheep were identified. The prevalence of the infection varied with the age of the animals

Serological diagnosis of chronic Chagas disease: Is it time for a change?

Chagas disease has spread to non-endemic areas 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, Architect Chagas (cut-off ≥ 1 S/CO, sample relative light units/cut-off value), as a single technique in the diagnosis of chronic Chagas disease. Architect Chagas showed a sensitivity of 100% (95% confidence interval, CI = 99.5-100) and a specificity of 97.6% (95% CI = 95.2-99.9). Five out of six false-positive sera were a consequence of cross-reactivity with Leishmania spp. and all of them achieved results < 5 S/CO. We propose Architect Chagas as a single technique for screening in blood banks and for routine diagnosis in clinical laboratories. Only grey zone and positive sera with a result ≤ 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 spp. 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

Serological diagnosis of chronic Chagas disease: Is it time for a change?

Chagas disease has spread to non-endemic areas 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, Architect Chagas (cut-off ≥ 1 S/CO, sample relative light units/cut-off value), as a single technique in the diagnosis of chronic Chagas disease. Architect Chagas showed a sensitivity of 100% (95% confidence interval, CI = 99.5-100) and a specificity of 97.6% (95% CI = 95.2-99.9). Five out of six false-positive sera were a consequence of cross-reactivity with Leishmania spp. and all of them achieved results < 5 S/CO. We propose Architect Chagas as a single technique for screening in blood banks and for routine diagnosis in clinical laboratories. Only grey zone and positive sera with a result ≤ 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 spp. 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