Comparison of four PCR methods for efficient detection of Trypanosoma cruzi in routine diagnostics

Abstract

Due to increased migration, Chagas disease has become an international health problem. Reliable diagnosis of chronically infected people is crucial for prevention of non-vectorial transmission as well as treatment. This study compared four distinct PCR methods for detection of Trypanosoma cruzi DNA for the use in well-equipped routine diagnostic laboratories. DNA was extracted of T. cruzi-positive and negative patients' blood samples and cultured T. cruzi, T. rangeli as well as Leishmania spp. One conventional and two real-time PCR methods targeting a repetitive Sat-DNA sequence as well as one conventional PCR method targeting the variable region of the kDNA minicircle were compared for sensitivity, intra- and interassay precision, limit of detection, specificity and cross-reactivity. Considering the performance, costs and ease of use, an algorithm for PCR-diagnosis of patients with a positive serology for T. cruzi antibodies was developed.

Detection and genotyping of Trypanosoma cruzi from açai products commercialized in Rio de Janeiro and Pará, Brazil

“…The selectivity of this set of primers could be explained by the high interspecific variability of the trypanosomatids sub-telomeric sequences used as targets. Thus, the primers Tc189F and Tc189R were used instead of primers that anneal to kinetoplast minicircle DNA (kDNA) and nuclear satellite DNA (satDNA) as those sets of primers may amplify DNA from T. rangeli [ 16 , 17 ].…”

Chagas disease: an overview of diagnosis

“…[74][75][76] Thus, several groups have implemented the usage of PCR (polymerase chain reaction) to identification of the genetic material from the parasite, in blood and serum samples as well as tissue samples. [77][78][79][80] Several types of the PCR techniques are available to detect T. cruzi DNA in serum and blood samples, among them we find: conventional PCR that amplifies a specific sequence from the parasite's DNA, for this it's common the use of repeated sequences or DNA from kinetoplast (kDNA). 77,81,82 Hot-Start PCR which is a modification to the conventional PCR to diminish amplification of unspecific products; 40 nested PCR is employed to amplify DNA sequences that are found in very low quantities in the parasite genome enhancing the sensitivity in the system, it consists in the extension of a specific sequence in two successive amplification steps.…”

“…[77][78][79][80] Several types of the PCR techniques are available to detect T. cruzi DNA in serum and blood samples, among them we find: conventional PCR that amplifies a specific sequence from the parasite's DNA, for this it's common the use of repeated sequences or DNA from kinetoplast (kDNA). 77,81,82 Hot-Start PCR which is a modification to the conventional PCR to diminish amplification of unspecific products; 40 nested PCR is employed to amplify DNA sequences that are found in very low quantities in the parasite genome enhancing the sensitivity in the system, it consists in the extension of a specific sequence in two successive amplification steps. 83 Several tools that utilize probes to verify presence/absence of specific DNA are used too (Southern Blot or PCR and hybridization) 84 Finally, real-time PCR usage allows to determine the parasite load by the quantification of the specific sequence amplification.…”

“…83 Several tools that utilize probes to verify presence/absence of specific DNA are used too (Southern Blot or PCR and hybridization) 84 Finally, real-time PCR usage allows to determine the parasite load by the quantification of the specific sequence amplification. 40,77,85 An important advantage that the use of PCR offers as a diagnosis tool is that allows the characterization of the circulating strains in an endemic area for Chagas disease. 86 Due to the wide genetic diversity in T. cruzi, the different strains of the parasite have been classified in six Discrete Typing Units (DTU).…”

Determinação da atividade biológica de bioterápico \'in vivo\' na infecção experimental determinada por Trypanosoma cruzi

“…Contudo, cada vez mais se discute a necessidade de um método mais mecânico, preciso e sensível, desta forma, pode ser aplicada técnicas moleculares como a reação de polimerase em cadeia em tempo real (qPCR), a qual possui boas performances, recomendando-se o seu uso na rotina laboratorial ( SCHIJMAN, 2018;SEIRINGER et al, 2017;VOLPATO et al, 2017;ZINGALES, 2017 e apresentaram uma redução da carga parasitária considerável (redução da carga parasitária, com relação ao controle, em 84%, 64% e 44% para os bioterápicos 30DH sangue FA, 30RC sangue pre e 30RC sangue, respectivamente).…”