Polymerase chain reaction detection: new insights into the diagnosis of chronic Chagas disease

Britto, Constança; Ma, Chang; Marques, Patrícia; Fernandes, Octávio; Cm, Morel

doi:10.1590/s0074-02761999000700056

Cited by 14 publications

(15 citation statements)

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“…A volume of 700 l was mixed with 1,400 l guanidine buffer, and the mixture was boiled 24 h later to avoid clot formation. DNA was extracted using the phenol-chloroform method (4,27) in a UV lightirradiated chamber under strict decontamination conditions. The amplification mixture was prepared in a different UV light-irradiated chamber and contained 3 mM MgCl 2 , 0.2 mM deoxynucleoside triphosphates, 0.1 M primers 121 (5Ј-AAA TAA TGT ACG GG(T/G) GAG ATG CAT GA-3Ј) and 122 (5Ј-GGT TCG ATT GGG GTT GGT GTA ATA TA-3Ј), 1.65 U GoTaq polymerase (Promega), and 10 l DNA.…”

Section: Experimental Designmentioning

confidence: 99%

Hydroxymethylnitrofurazone Is Active in a Murine Model of Chagas' Disease

Davies

Cardozo

Negrette

et al. 2010

Antimicrob Agents Chemother

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The addition of a hydroxymethyl group to the antimicrobial drug nitrofurazone generated hydroxymethylnitrofurazone (NFOH), which had reduced toxicity when its activity against Trypanosoma cruzi was tested in a murine model of Chagas' disease. Four groups of 12 Swiss female mice each received 150 mg of body weight/kg/day of NFOH, 150 mg/kg/day of nitrofurazone (parental compound), 60 mg/kg/day of benznidazole (BZL), or the solvent as a placebo. Treatments were administered orally once a day 6 days a week until the completion of 60 doses. NFOH was as effective as BZL in keeping direct parasitemia at undetectable levels, and PCR results were negative. No histopathological lesions were seen 180 days after completion of the treatments, a time when the levels of anti-T. cruzi antibodies were very low in mice treated with either NFOH or BZL. Nitrofurazone was highly toxic, which led to an overall rate of mortality of 75% and necessitated interruption of the treatment. In contrast, the group treated with its hydroxymethyl derivative, NFOH, displayed the lowest mortality (16%), followed by the BZL (33%) and placebo (66%) groups. The findings of histopathological studies were consistent with these results, with the placebo group showing the most severe parasite infiltrates in skeletal muscle and heart tissue and the NFOH group showing the lowest. The present evidence suggests that NFOH is a promising anti-T. cruzi agent.In 1909, Carlos Chagas described the protozoan parasite Trypanosoma cruzi as the causative agent of a widespread disease in Brazil that involved cardiac alterations and megaorgans. The parasite and its insect vectors were described in the original paper on the disease (7), and the parasite was soon confirmed to be present in patients throughout Latin America. Recent reports estimate the number of infected people to be between 9.8 million and 15 million in the Americas (29), with 28 million more being at risk of infection (26). In this scenario, congenital transmission of infection is a major problem in urban areas, along with risks from blood transfusion and migration of infected patients to areas of nonendemicity (28). In spite of the reduction in the number of new infections and the increased knowledge about the parasite, treatment of Chagas' disease relies on two drugs developed during the early 1970s: benznidazole (BNZ; Radanil, Roche) and nifurtimox (NFX; Bayer) (35). Both drugs are generally well tolerated by children but cause many undesirable side effects in adults and are not effective during the chronic phase of the disease. A well-tolerated, safe, and therapeutically efficient drug is not yet available. Several attempts have been made to create new drugs with more specificity toward the parasite and less toxicity for the mammalian host. Such attempts included inhibitors of cruzain (13, 14), C-14␣-demethylase (5, 10, 32), and chemical modifications of molecules with known trypanocidal activity, such as aromatic nitroheterocyclic compounds (1, 2). Nitrofurazone is an antimicrobial drug that is eff...

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Section: Experimental Designmentioning

confidence: 99%

Hydroxymethylnitrofurazone Is Active in a Murine Model of Chagas' Disease

Davies

Cardozo

Negrette

et al. 2010

Antimicrob Agents Chemother

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“…Actual quantitation of T. cruzi DNA using real-time PCR can be used to generate information related to the progression of the disease, and to correlate parasite density and distribution with the condition of the animal at the time of death. 48,56,57 We are aware of a limitation of our study, namely the absence of immunologic tests in the same animals; however, in our previous work with archived baboon samples we showed that the presence of parasites in the tissue and not seropositivity per se correlated positively with amount of DNA detected. 39…”

Section: Discussionmentioning

confidence: 88%

Polymerase Chain Reaction Detection of Trypanosoma cruzi in Macaca fascicularis Using Archived Tissues

Williams¹,

Mubiru²,

Schlabritz‐Loutsevitch³

et al. 2009

The American Journal of Tropical Medicine and Hygiene

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This study describes conventional and real-time polymerase chain reaction (PCR) methods developed to detect and quantify Trypanosoma cruzi DNA in cynomolgus monkeys (Macaca fascicularis) using formalin-fixed paraffin-embedded blocks archived for periods of up to 6 years. The highest concentration of T. cruzi DNA was found in the myocardium, urinary bladder, stomach, lymph node, adrenal gland, and colon. The concentration of T. cruzi DNA detected in cardiac tissues was 10-100-fold greater than found elsewhere; the mean concentrations of T. cruzi DNA in non-cardiac tissues were otherwise comparable. Trypanosoma cruzi DNA was amplified from cerebrum but not cerebellum or kidney. Successful use of DNA from formalin-fixed, paraffin-embedded blocks is important because most pathology laboratories routinely archive wax blocks. This archived resource can be used for further studies on the prevalence of this disease.

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“…dimidiata investigated by Dorn et al (1999), more than twice as many were found positive by PCR as by microscopy. When Britto et al (1999) used XD-M and PCR-based tests of blood samples to test subjects from four endemic areas of Brazil, the XD-M (with 24.5%, 13.0%, 32.4%, and 2.4% of subjects from each area positive) always appeared much less sensitive than the direct PCR (with corresponding percentages of 96.5%, FIG. The amplicons produced, in the PCR, from faecal samples of Triatoma infestans that had fed on four cases (1, 2, 3 and 4) of chronic American trypanosomiasis 30, 60 or 90 days earlier (a), and the corresponding Southern blots in which the P 32 -labelled kinetoplastid DNA of Trypanosoma cruzi was used as a probe (b).…”

Section: Discussionmentioning

confidence: 99%

The PCR-based detection ofTrypanosoma cruziin the faeces ofTriatoma infestansfed on patients with chronic American trypanosomiasis gives higher sensitivity and a quicker result than routine xenodiagnosis

Zulantay

Apt

Gil

et al. 2007

Annals of Tropical Medicine & Parasitology

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In the xenodiagnosis (XD) of American trypanosomiasis (Chagas disease), Trypanosoma cruzi in the triatomine bugs fed on the patient can now be detected using PCR (XD-PCR) as well as by microscopy (XD-M). In a study to compare XD-PCR with XD-M, triatomine bugs were fed on 50 cases of chronic American trypanosomiasis, of whom only 25 were ever found positive by XD-M. Overall, the bugs fed on 34 of the patients (all 25 cases found positive by XD-M and nine of the other patients) were found PCR-positive, giving a 330-bp fragment corresponding to part of the hyper variable region of the kinetoplast DNA of T. cruzi. Of the 25 patients who were ever found positive by XD-M, 20 gave bugs that were smear-positive on day 90 and a similar number (24; P=0.125) gave bugs that were PCR-positive at this time. On day 30, however, the bugs fed on only 11 of these 25 patients were found positive by microscopy, whereas 23 of these patients were found positive by XD-PCR (P=0.0016). Thus, not only was XD-PCR more sensitive than XD-M but it was also quicker, revealing more cases within 30 days than detected using XD-M over a period of 90 days.

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Polymerase chain reaction detection: new insights into the diagnosis of chronic Chagas disease

Cited by 14 publications

References 0 publications

Hydroxymethylnitrofurazone Is Active in a Murine Model of Chagas' Disease

Hydroxymethylnitrofurazone Is Active in a Murine Model of Chagas' Disease

Polymerase Chain Reaction Detection of Trypanosoma cruzi in Macaca fascicularis Using Archived Tissues

The PCR-based detection ofTrypanosoma cruziin the faeces ofTriatoma infestansfed on patients with chronic American trypanosomiasis gives higher sensitivity and a quicker result than routine xenodiagnosis

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