Sensitivity of polymerase chain reaction for detection of known aliquots of Trypanosoma cruzi in the blood of mice: an in vitro study

Abstract

To evaluate the sensitivity of polymerase chain reaction (PCR) to reveal known number of trypomastigote in the blood of mice, three separate experiments were done. First: To eight samples of 500mul of normal mice blood, one aliquot of 1, 2, 3, 4, 5, 10, and 50 trypomastigotes respectively, were added. Second and third: 10 aliquots with 1 and 10 with 2 trypomastigotes were added to samples of 500mul of normal mice blood. Positive control: 500mul of blood containing 100,000 trypomastigotes. For kDNA minicircles amplification by PCR the primers:S35 and S36 were used. PCR revealed products of 330 b.p in the positive controls. When only one sample with the aliquots of 1 or 2 trypomastigotes was examined, results were negative; results were positive with aliquots of 3 to 50 trypomastigotes. In the 2nd and 3rd experiments, 9/10 aliquots with one parasite and 9/10 with 2 trypomastigotes were positive revealing a high sensitivity of this reaction. In conclusion, the presence of one single parasite in 500mul of blood, is enough for a positive PCR. This method could be used as a complement to the various parasitological cure tests in treated mice, when low volumes of blood are individually examined.

The detection ofTrypanosoma cruziby nested-PCR in elderly patients: relationship to the clinical and epidemiological profile

“…37 Piron et al 38 reported that qPCR (quantitative real-time PCR) was sensitive to 0.8 parasites/ml. Campos et al 39 reported that a PCR for kDNA detection in mice was able to detect 2-4 parasites/ml. The latter study also showed that for smaller numbers of parasites the assay (extraction and PCR) should be done several times to confirm the result.…”

Trypanosoma cruzi: susceptibility to chemotherapy with benznidazole of clones isolated from the highly resistant Colombian strain

“…These findings point to a difficulty in the use of PCR as a cure test in mice, considering the small volume of blood sample obtained from each mouse, resulting in the low sensitivity observed in the present study. A previous study by Campos et al 17 investigated the sensitivity of PCR to reveal the presence of known aliquots of trypomastigotes added to the blood of normal mice in vitro. The findings indicated a high sensitivity of PCR to detect even one trypomastigote in the blood, but examination of several samples may be necessary.…”

“…This test has been recently included for the diagnosis of chronic infection in humans 10 11 12 and as a cure test in treated patients 13 . An evaluation of the sensitivity of this test has been performed in the blood containing known aliquots of trypomastigotes 17 . However, the sensitivity of PCR for post-treatment detection of sub-patent parasitemia in mice has not been previously evaluated, in comparison with the standard tests.…”

Diagnosis of animal trypanosomoses: proper use of current tools and future prospects

“…PCR improved the sensitivity for detecting active infections and significantly improved specificity, at various taxonomic levels. However, molecular methods have critical limitations: (i) they leave cases with low parasitaemia or non-circulating parasites undetected [ 31 , 32 ]; (ii) they are limited to fully equipped laboratories with skilled technicians; (iii) positive results are conclusive of active infection (leaving aside the fact that DNA may still be detected 24–48 h after curative treatment [ 33 ]), but negative results are not; and (iv) there is a significant delay between the time of sampling in the laboratory and the delivery of results, so animals can be out of reach by the time the veterinarian, vet technician or owner receives the results. Loop-mediated isothermal amplification methods (LAMP) applied to parasite DNA can mitigate such drawbacks.…”