Use of a nested polymerase chain reaction (N-PCR) to detect Trypanosoma cruzi in blood samples from chronic chagasic patients and patients with doubtful serologies

Marcon, Gláucia Elisete Barbosa; Andrade, Paula Durante; Albuquerque, Dulcinéia Martins; Wanderley, Jamiro da Silva; Almeida, Eros Antônio de; Guariento, Maria Elena; Costa, Sandra Cecı́lia Botelho

doi:10.1016/s0732-8893(02)00366-8

Cited by 68 publications

(60 citation statements)

References 20 publications

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“…30,31 Our results are consistent with those of a previous report that indicate that the sensitivity of the Tcz1/ Tcz2 PCR is approximately 10 times higher than that of the PCR using the kS35Ј/S36Ј primers, 6 despite the theoretically similar number of targets. Such results might be related to an incomplete dispersion of the dense kinetoplast minicircle present in the blood samples.…”

Section: Discussionsupporting

confidence: 92%

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

Virreira

Torrico

Truyens

et al. 2003

The American Journal of Tropical Medicine and Hygiene

156

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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.

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Section: Discussionsupporting

confidence: 92%

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

Virreira

Torrico

Truyens

et al. 2003

The American Journal of Tropical Medicine and Hygiene

156

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“…Although some authors have demonstrated that PCR is more sensitive than hemoculture and xenodiagnosis (6), its sensitivity is not 100% due to the fact that detecting a parasite in 10 ml of blood is slightly complicated in terms of steric hindrance and the availability of template DNA within the same reaction mixture. Some authors have also reported the possibility of using a nested PCR in order to increase the amount of parasite template DNA (22).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Adult Chronic Chagas' Heart Disease Diagnosis by Molecular and Serological Methods

et al. 2009

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Chagas' disease caused by Trypanosoma cruzi is endemic in Latin America. T. cruzi presents heterogeneous populations and comprises two main genetic lineages, named T. cruzi I and T. cruzi II. Diagnosis in the chronic phase is based on conventional serological tests, including indirect immunofluorescence (IIF) and enzymelinked immunosorbent assay (ELISA), and diagnosis in the acute phase based on parasitological methods, including hemoculture. The objective of this study was to evaluate the diagnostic procedures of Chagas' disease in adult patients in the chronic phase by using a PCR assay and conventional serological tests, including TESA-blot as the gold standard. Samples were obtained from 240 clinical chronic chagasic patients. The sensitivities, compared to that of TESA-blot, were 70% for PCR using the kinetoplast region, 75% for PCR using the nuclear repetitive region, 99% for IIF, and 95% for ELISA. According to the serological tests results, we recommend that researchers assess the reliability and sensitivity of the commercial kit Chagatest ELISA recombinant, version 3.0 (Chagatest Rec v3.0; Wiener Lab, Rosario, Argentina), due to the lack of sensitivity. Based on our analysis, we concluded that PCR cannot be validated as a conventional diagnostic technique for Chagas' disease. These data have been corroborated by low levels of concordance with serology test results. It is recommended that PCR be used only for alternative diagnostic support. Using the nuclear repetitive region of T. cruzi, PCR could also be applicable for monitoring patients receiving etiologic treatment.

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“…NPCR -The NPCR was performed as previously described by our group (Marcon et al 2002) with some modifications. The first reaction was performed with 2 µL of the DNA sample, 50 mM of potassium chloride, 10 mM of Tris-HCl (pH 8.4), 2.5 mM magnesium chloride, 2.0 mM of each oligonucleotide (TCZ1 and TCZ2 -first reaction; TCZ3 and TCZ4 -second reaction) described in Table II, deoxyribonucleic mixture (1 mM) -dNTPs (dATP, dGTP, dCTP; dTTP) and 2.0 U of Taq DNA polymerase (Invitrogen) in a final volume of 30 µL.…”

Section: Patients Materials and Methodsmentioning

confidence: 99%

“…It was shown that the detected DNA is derived from the persistence of the parasite in the affected tissues and not from DNA persistence over long periods of time. Piron et al (2007) used nested PCR (NPCR) and real-time techniques to detect and quantify T. cruzi in blood samples of chagasic patients, obtaining a similar positivity rate to results obtained by Marcon et al (2002). The real-time PCR technique was also employed to detect T. cruzi in the amniotic fluid of mothers with Chagas disease (Virreira et al 2006) and to subtype T. cruzi I and II in infected tissues by means of the difference in primer annealing temperatures.…”

mentioning

confidence: 88%

Trypanosoma cruzi: parasite persistence in tissues in chronic chagasic Brazilian patients

Marcon

Albuquerque

Batista

et al. 2011

Mem. Inst. Oswaldo Cruz

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Use of a nested polymerase chain reaction (N-PCR) to detect Trypanosoma cruzi in blood samples from chronic chagasic patients and patients with doubtful serologies

Cited by 68 publications

References 20 publications

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

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

Evaluation of Adult Chronic Chagas' Heart Disease Diagnosis by Molecular and Serological Methods

Trypanosoma cruzi: parasite persistence in tissues in chronic chagasic Brazilian patients

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