Comparison of Polymerase Chain Reaction Methods for Reliable and Easy Detection of Congenital Trypanosoma Cruzi Infection

Abstract

Abstract. The polymerase chain reaction (PCR) is a potentially interesting diagnostic tool for detecting congenital Trypanosoma cruzi infection at birth. We have compared the sensitivity and capacity of a group of T. cruzi PCR primers in detecting the complete spectrum of known T. cruzi lineages, and to improve and simplify the detection of infection in neonatal blood. We found that the two primers, Tcz1/Tcz2 and Diaz1/Diaz2, which target the 195-basepair satellite repeat, detected all parasitic lineages with the same sensitivity. However, the intensity of the amplicon was somewhat higher with Tcz1/Tcz2. For other tested primers (nuclear DNA primers BP1/BP2, O1/O2, Pon1/Pon2, and Tca1/Tca2 and kinetoplast DNA primers S35Ј/S36Ј and 121/122), either the intensity of amplicons varied according to T. cruzi lineages or the PCR assay was less sensitive. The use of the Tcz1/Tcz2 primers, which target a tandem repetitive sequence, requires a careful determination of the appropriate amount of Taq polymerase to avoid the formation of smears and multiple amplicon bands. The Tcz1/Tcz2 primers resulted in an intense 200-basepair amplicon with DNA extracted from blood equivalent to 0.02 parasites per assay when used with a simple DNA extraction method and of a low amount of Taq polymerase from a standard PCR kit. To better assess such PCR protocol, we assayed 311 samples of neonatal blood previously tested by parasitologic methods. The reliability of our PCR test was demonstrated, since all the 18 blood samples from newborns with congenital T. cruzi infection were positive, whereas the remaining samples (30 from control newborns of uninfected mothers and 262 of 263 from babies born to infected mothers) were negative. Since our PCR method is simple, reliable, robust, and inexpensive, it appears suitable for the detection of T. cruzi infection in neonatal blood, even in laboratories that are not equipped for performing the PCR.

Caracterización biológica y genética de dos clones pertenecientes a los grupos I y II de Trypanosoma cruzi de Colombia

“…Los productos amplificados fueron analizados en geles de agarosa al 2%, teñidos con bromuro de etidio y visualizados bajo un transiluminador UV (29). Cada uno de estos ensayos se hizo por duplicado.…”

Usefulness of PCR-based assays to assess drug efficacy in Chagas disease chemotherapy: value and limitations

“…In general, the true potential of real-time PCR will be recognized in situations for which PCR-based techniques have been promoted, such as congenital infections (Schijman et al 2003, Virreira et al 2003, Mora et al 2005, monitoring parasitaemia during and after treatment (Russomando et al 1998, Britto et al 2001, Schijman et al 2003, early detection of relapses after heart transplantation (Maldonado et al 2004) and other immunosuppressive circumstances. To deal with more precise results and make them comparable between laboratories, the main constraint until now has been the lack of a universal reagent presenting accurately quantified T. cruzi DNA samples to be used as standards in all quantification assays (Piron et al 2007).…”

Mother-to-Child Transmission of Trypanosoma cruzi

“…This strategy is a major challenge in the field: direct parasitological examinations require trained personnel and have a low sensitivity, and loss to follow-up often precludes serological examinations after 8 months. Polymerase chain reaction molecular techniques have also been used along with other serological and or parasitological assays to diagnose congenital T cruzi [10][11][12].…”