Use of a Simplified Polymerase Chain Reaction Procedure to Detect Trypanosoma cruzi in Blood Samples from Chronic Chagasic Patients in a Rural Endemic Area

Wincker, Patrick; Britto, Constança; Pereira, João Baptista Borges; Cardoso, Maria Angélica; Oelemann, Walter; Morel, Carlos Médicis

doi:10.4269/ajtmh.1994.51.771

Cited by 256 publications

(199 citation statements)

References 8 publications

Supporting

Mentioning

175

Contrasting

Unclassified

Order By: Relevance

“…39 A PCR targeting the kinetoplast DNA of T. cruzi was performed as previously described using primers 121 (5=-AAATAATGTACGGGKGAGATGCATGA-3=) and 122 (5=-GGTTCGATTGGGGTTGGTGTAATATA-3=). 40,41 We included internal control primers that amplify a 71-bp fragment. This fragment corresponds to multicopy nuclear DNA short interspersed element that is specific to guinea pigs.…”

Section: Dna Extraction and Pcrmentioning

confidence: 99%

Cavia porcellus as a Model for Experimental Infection by Trypanosoma cruzi

Castro-Sesquen

Gilman

Yauri

et al. 2011

The American Journal of Pathology

View full text Add to dashboard Cite

The guinea pig (Cavia porcellus) is a natural reservoir for Trypanosoma cruzi but has seldom been used as an experimental infection model. We developed a guinea pig infection model for acute and chronic Chagas disease. Seventy-two guinea pigs were inoculated intradermally with 10 4 trypomastigotes of T. cruzi strain Y (experimental group); 18 guinea pigs were used as control group. Eight animals from the experimental group and two from the control group were Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is one of the most important parasitic infections of the Americas. Despite successful vector control programs in many countries, the disease remains a major public health problem in Latin America, with 8 to 10 million people currently infected, an annual incidence of 65,000 new cases in 15 countries, and 14,000 deaths associated with the infection per year. 1 Transmission to the mammalian host occurs when a T. cruzi-infected triatomine vector deposits infectious trypomastigotes in feces during a blood meal and the parasites invade through the bite wound or intact mucosal membranes. 2 Trypomastigotes can infect virtually any nucleated cell, where they transform to amastigotes and replicate in the host cell cytoplasm. Amastigotes convert back to trypomastigotes inside the cell and then rupture the cell and circulate in the bloodstream. After weeks, the host immune response usually controls the acute infection but does not completely clear the parasite. This results in lifelong chronic infection of the host.The initial weeks after T. cruzi infection are characterized by a high-level parasitemia and symptoms that vary from mild nonspecific febrile illness to life-threatening myocarditis and/or meningoencephalitis. 2 Acute symptoms and detectable parasitemia resolve spontaneously within 2 to 3 months, and the infected individual passes into the chronic phase of the disease. Nearly all individuals in the early period of chronic infection are asymptomatic and are said to have the indeterminate form of the disease. In most individuals, the infection remains silent for life and is detectable only by anti-T. cruzi serologic tests and, in a proportion of individuals, by molecular Supported by NIH training grant in infectious and tropical diseases 5 T35 AI065385 and NIH grant 1R01AI087776-01.

show abstract

Section: Dna Extraction and Pcrmentioning

confidence: 99%

Cavia porcellus as a Model for Experimental Infection by Trypanosoma cruzi

Castro-Sesquen

Gilman

Yauri

et al. 2011

The American Journal of Pathology

View full text Add to dashboard Cite

show abstract

“…Inasmuch a cross reaction with T. rangeli was present. As previously referred, Vargas et al 18 , through different dilutions has done an indirect evaluation of the sensitivity of PCR in mice.…”

Section: Resultsmentioning

confidence: 99%

“…Several primers have been used for the identification of fragments with different base pairs. Experimentally, Vargas et al 18 , testing different dilutions of blood of infected mice determined the positivity of PCR by the identification of fragments of 270 b.p until a dilution of 1x10 -2 parasites/ml. However these data did not establish the exact number of parasites in each sample nor clarified the number of samples to examine in order to obtain such results.…”

mentioning

confidence: 99%

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

Campos

Magalhães

Reis

et al. 2002

Rev. Soc. Bras. Med. Trop.

View full text Add to dashboard Cite

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.

show abstract

“…Bearing this in mind, in this work we evaluated the use of TcH2AF/ R 20 and TrF/R2 PCRs 18 to identify these trypanosomes by comparing them with conventional techniques and with the S35/S36 PCR, the most sensitive PCR described for T. cruzi 3,4,40,41 in the two last decades. Despite the S35/S36 PCR being described as a specific tool for the amplification of kDNA from T. cruzi at the beginning 8,29 , sequencing of kDNA minicircles of T. rangeli found a high degree of homology among conserved regions between the two species.…”

Section: Discussionmentioning

confidence: 99%

“…Due to direct microscopic detection of trypanosomes, the traditional method for assessment of infection in vectors is not able to distinguish T. cruzi from T. rangeli infection, several polymerase chain reaction techniques have been developed 3,4,5,6,9,13,19,28,33,36,40,41 . However, current PCR assays used for mixed infection detection show some disadvantages such as the amplification of bands of similar size both in T. cruzi and T. rangeli 27,28 , the amplification of polymorphic fragments 11,19 , and bias to T. cruzi in the case of mixed T. cruzi and T. rangeli infection 9,33,36 .…”

Section: Introductionmentioning

confidence: 99%

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes

Pavía

Vallejo

Montilla

et al. 2007

Rev. Inst. Med. trop. S. Paulo

View full text Add to dashboard Cite

SUMMARYTrypanosoma rangeli is non pathogenic for humans but of important medical and epidemiological interest because it shares vertebrate hosts, insect vectors, reservoirs and geographic areas with T. cruzi, the etiological agent of Chagas disease. Therefore, in this work, we set up two PCR reactions, TcH2AF/R and TrFR2, to distinguish T. cruzi from T. rangeli in mixed infections of vectors based on amplification of the histone H2A/SIRE and the small nucleolar RNA Cl1 genes, respectively. Both PCRs were able to appropriately detect all T. cruzi or T. rangeli experimentally infected-triatomines, as well as the S35/S36 PCR which amplifies the variable region of minicircle kDNA of T. cruzi. In mixed infections, whereas T. cruzi DNA was amplified in 100% of samples with TcH2AF/R and S35/S36 PCRs, T. rangeli was detected in 71% with TrF/R2 and in 6% with S35/S36. In a group of Rhodnius colombiensis collected from Coyaima (Colombia), T. cruzi was identified in 100% with both PCRs and T. rangeli in 14% with TrF/R2 and 10% with S35/S36 PCR. These results show that TcH2AF/R and TrF/R2 PCRs which are capable of recognizing all T. cruzi and T. rangeli strains and lineages could be useful for diagnosis as well as for epidemiological field studies of T. cruzi and T. rangeli vector infections.

show abstract

Use of a Simplified Polymerase Chain Reaction Procedure to Detect Trypanosoma cruzi in Blood Samples from Chronic Chagasic Patients in a Rural Endemic Area

Cited by 256 publications

References 8 publications

Cavia porcellus as a Model for Experimental Infection by Trypanosoma cruzi

Cavia porcellus as a Model for Experimental Infection by Trypanosoma cruzi

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

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes

Contact Info

Product

Resources

About