Trypanosoma cruzi: specific detection of parasites by PCR in infected humans and vectors using a set of primers (BP1/BP2) targeted to a nuclear DNA sequence.

In the present work we evaluate Trypanosoma cruzi DNA detection by PCR using the nuclear oligonucleotides BP1/BP2 as primers. These primers are targeted to the 5' and 3' ends of the coding region for the flagellar protein F29. An amplification product of BP1/BP2 is a DNA band 692 bp long. Titration assays were performed to evaluate the minimum amount of parasite DNA that can be detected by this assay, resulting in 10 fg (equivalent to about 1/20 of the genome). The assay was also performed using T. cruzi DNA from different strains, clones, and human-derived isolates obtaining, in all cases, amplification products. No DNA amplification was observed when the PCR was performed using DNA from Leishmania braziliensis, but when T. rangeli DNA was used, a 615-bp-long fragment was amplified. Under appropriate gel conditions T. cruzi and T. rangeli DNA amplicons could be differentiated. When both conventional xenodiagnosis and PCR detection of parasite DNA in the feces of insect vectors fed with blood from infected patients were compared, 10 of 20 samples were positive by both techniques. However, 2 other samples with positive serology were also positive by PCR. When PCR was performed on blood samples from infected and uninfected individuals, 62 of 65 serologically positive human samples amplified the BP1/BP2 692-bp T. cruzi DNA fragment (sensitivity >95%). The 3 negative samples were positive when Southern blot hybridization was performed using the radiolabeled PCR amplification product as probe (sensitivity 100%).

Silber AM ，Búa J ，Porcel BM ，Segura EL ，Ruiz AM ... -  《EXPERIMENTAL PARASITOLOGY》

Amplification of a specific repetitive DNA sequence for Trypanosoma rangeli identification and its potential application in epidemiological investigations.

Trypanosoma rangeli can infect humans as well as the same domestic and wild animals and triatomine vectors infected by Trypanosoma cruzi in Central and South America. This overlapping distribution complicates the epidemiology of American trypanosomiasis due to the cross-reactivity between T. rangeli and T. cruzi antigens and the presence of conserved DNA sequences in these parasites. We have isolated a T. rangeli-specific DNA repetitive element which is represented in approximately 103 copies per parasite genome and is distributed in several chromosomal bands. The 542-bp nucleotide sequence of this element, named P542, was determined and a PCR assay was standardized for its amplification. The sensitivity of the assay is high, allowing the detection of one tenth of the DNA content of a single parasite. The presence of the P542 element was confirmed in 11 T. rangeli isolates from mammalian hosts and insect vectors originating from several countries in Latin America. Negative amplification was observed with different T. cruzi strains and other trypanosomatids. The potential field application of the P542 PCR assay was investigated in simulated samples containing T. rangeli and/or T. cruzi and intestinal tract and feces of Rhodnius prolixus. Epidemiological studies were conducted in DNA preparations obtained from the digestive tracts of 12 Rhodnius colombiensis insects collected in a sylvatic area in Colombia. Positive amplification of the P542 element was obtained in 9/12 insects. We have also compared in the same samples the diagnostic performance of two PCR assays for the amplification of the variable domain of minicircle kinetoplast DNA (kDNA) and of the large subunit (LSU) of the ribosomal RNA gene of T. cruzi and T. rangeli. Data indicate that the kDNA PCR assay does not allow diagnosis of mixed infections in most insects. On the other hand, the PCR assay of the LSU RNA gene showed lower sensitivity in the detection of T. rangeli than the PCR assay of the P542 element. It is predicted that the use of sensitive detection techniques will indicate that the actual distribution of T. rangeli in America is wider than presumed.

Vargas N ，Souto RP ，Carranza JC ，Vallejo GA ，Zingales B ... -  《EXPERIMENTAL PARASITOLOGY》

Trypanosoma cruzi: parasite detection and strain discrimination in chronic chagasic patients from northeastern Brazil using PCR amplification of kinetoplast DNA and nonradioactive hybridization.

Blood samples from 172 individuals from northeastern Brazil were subjected to PCR amplification of Trypanosoma cruzi-specific kDNA sequences. This method enabled us to detect parasite DNA in 21 of 47 patients that were serologically positive. In addition, 1 patient that gave doubtful results with chagasic serology was confirmed as positive by PCR. We applied the same PCR detection method to the feces of wild triatomines captured in the same region, obtaining three positive results that were confirmed by microscopic examination. The 25 amplified products obtained in this study were then reamplified with primers that gave a final amplicon containing sequences from the most variable region of kDNA minicircles. These were used as probes in hybridization experiments aimed at defining the degree of relatedness between the strains infecting humans and insects based on kDNA homologies. We found that the amplification products from the three triatomines were related and showed no cross-hybridization with those obtained from human infections. Eight amplified products from human infections showed no cross-hybridization and did not hybridize with products from other patients. This indicates that the strains of T. cruzi circulating in the region present a high level of genetic heterogeneity. Finally, a number of amplified products hybridized with amplicons that did not hybridize with each other, indicating that infections with a parasite population presenting a mixed kDNA content (either due to different strains of T. cruzi or to a hybrid parasite) are a more frequent event than previously thought.

Britto C ，Cardoso MA ，Ravel C ，Santoro A ，Pereira JB ，Coura JR ，Morel CM ，Wincker P ... -  《EXPERIMENTAL PARASITOLOGY》

[Comparison of PCR methods for the diagnosis of congenital Trypanosoma cruzi infection].

PCR is a potentially interesting diagnostic tool to detect congenital T. cruzi infection. We have compared the sensitivity and capacity of a battery of T. cruzi PCR primers to detect the complete spectrum of known T. cruzi lineages, in order to improve and simplify the detection of infection in neonatal blood. We found that the primers Tcz1/Tcz2, targeting the 195 bp satellite repeat, detected all the parasitic lineages with the same sensitivity For all other tested primers (nDNA primers: BP1/BP2, 01/02, Pon1/ Pon2 and Tca1/Tca2; kDNA primers: S35VS36, 121/122), either, the intensity of amplicons varied according to T. cruzi lineages, or the assess were less sensitive. In order to better assess such PCR protocol, we assayed 311 samples of neonatal blood previously tested with parasitological methods. 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 out of 263 from babies, parasitologically negative, born from infected mothers) were negative. As our PCR method is simple, reliable, robust and cheap, it appears suitable for the detection of T. cruzi infection in neonatal blood.

Virreira M ，Torrico F ，Truyens C ，Alonso-Vega C ，Solano M ，Carlier Y ，Svoboda M ... -  《Revista da Sociedade Brasileira de Medicina Tropical》

Trypanosoma cruzi: polymerase chain reaction-based detection in dried feces of Triatoma infestans.

PCR was employed to detect Trypanosoma cruzi DNA in Triatoma infestans dry fecal spots collected on filter papers. Both insects fed on experimentally infected monkeys and insects collected in a Paraguayan endemic area for Chagas' disease were examined. When the insects fed on a chronically infected monkey with low parasitemia as revealed by direct microscopic observation (DMO), T. cruzi was detected in the insect feces by PCR as soon as 2 days postfeeding. When the same experiment was performed on monkeys with parasitemia levels below the limit of detection by DMO, the degree of positivity found through PCR-Southern hybridization, applied on Day 8 postfeeding, was superior to that obtained through xenoculture. These results suggest that PCR can be used to speed the xenodiagnosis results with great sensitivity. On the other hand, when applied to the feces of triatomines collected in the field, 84% were positive by PCR-Southern hybridization, whereas only 26% were positive by DMO. Therefore, PCR could also be applied to the monitoring of the infection status of triatomines which infest rural dwellings by examining only the feces left on paper sensors hung on the walls of the houses.

Russomando G ，Rojas de Arias A ，Almiron M ，Figueredo A ，Ferreira ME ，Morita K ... -  《EXPERIMENTAL PARASITOLOGY》