Polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients.

The feasibility of DNA amplification by the polymerase chain reaction for specific detection of Trypanosoma cruzi in human blood was investigated. We have used primers flanking a 220-bp fragment of highly repetitive elements, the E13 element, in T. cruzi nuclear DNA. Only polymerase chain reaction products from blood samples of chronic chagasic patients showed several amplified fragments in 1.6% agarose gels stained with ethidium bromide. Southern blot analysis demonstrated that the 220-bp amplified fragment is specific for T. cruzi DNA and very useful to detect the presence of the parasite in blood from chronic chagasic patients.

Trypanosoma cruzi: changes in lipid composition during aging in culture.

Changes in lipid composition and fatty acid distribution in lipid fractions from total extracts of Trypanosoma cruzi were studied in culture from Day 2 to Day 14. This comprises the phases of exponential, stationary, and declining growth. Total phospholipid content decreased steadily during the three culture phases due to the marked reduction of phosphatidylcholine. Phosphatidylethanolamine increased during the exponential and declining phases. Thus, the final phosphatidylethanolamine/phosphatidylcholine ratio was higher than that determined on the second day. Sterols and acylglycerides increased as cultures aged. Fatty acid composition of different fractions varied during aging: phosphatidylcholine and phosphatidylethanolamine presented an increase of saturated and reduction of polyunsaturated (linoleic) acids, while for lysophosphatidylcholine and acylglycerides, the opposite change occurred. The modifications described may produce reduction of membrane fluidity and indicate that lipids participate actively in the adaptation of T. cruzi to the environmental changes produced by aging in culture.

Trypanosoma cruzi: involvement of proteolytic activity during cell fusion induced by epimastigote form.

Human erythrocytes were fused by Trypanosoma cruzi from 7 and 14 day old culture (stationary and declination phases, respectively) while only lysis was induced by 4 day old culture parasite (exponential phase). Lysis and erythrocyte fusion were studied by phase contrast microscopy, measuring of hemolysis and gel electrophoresis. The fusogenicity is Ca2+-dependent while lysis is delayed in the absence of exogenous Ca2+. The proteolysis of erythrocyte protein bands 1, 2, 2.1, 2.3 and 3 are common features of both fusion and lysis processes. Nevertheless the breakdown rate of ankyrin (band 2.1) and band 3 are different in fused or in lysed cells. The lysis process is associated with a faster degradation of band 2.1 and increase of band 2.3 than in the case of the fusion process. By contrast, degradation of band 3 occurs faster in the fusion than in the lytic event. Treatment of fusogenic parasites but not erythrocytes with TPCK, soybean trypsin inhibitor or FCS inhibited to some extent the fusion process and the decrease of bands 1, 2, 2.1, 2.3 and 3. The results suggest that proteases from fusogenic parasites may be directly or indirectly involved in the proteolysis of band 2.1 in a way related to induction of fusion.

Risk progression to chronic Chagas cardiomyopathy: influence of male sex and of parasitaemia detected by polymerase chain reaction.

BACKGROUND: Polymerase chain reaction (PCR) allows detection of Trypanosoma cruzi in blood throughout the course of Chagas' disease. OBJECTIVE: To determine whether T cruzi DNA detected by PCR is associated with progression to chronic Chagas cardiomyopathy. DESIGN: Prospective cohort study. SETTING: A tertiary care centre in Argentina. PATIENTS: 56 consecutive patients with chronic T cruzi infection. METHODS: Clinical examination, ECG, and Doppler echocardiography were carried out at baseline and at the end of the follow up. Detection of T cruzi DNA by PCR amplifying a nuclear sequence was undertaken in all patients at baseline. MAIN OUTCOME MEASURES: Progression was defined as death from chronic cardiomyopathy or the presence of a new ECG or left ventricular echocardiographic abnormality at the end of follow up. RESULTS: The 56 patients (21 male, 35 female; mean (SD) age, 56.0 (11.3) years) were followed for a mean 936.3 (244.39) days. Progression to cardiomyopathy was detected in 12 patients (21.4%). Three of these patients died after baseline evaluation. Univariate analysis showed that a positive PCR (relative risk 4.09, 95% confidence interval (CI) 1.60 to 9.85) and male sex (5.00, 95% CI 1.65 to 15.73) were associated with progression. Multivariable logistic regression indicated that both sex and PCR were independent variables affecting the outcome. CONCLUSIONS: In a cohort of seropositive individuals, patients with T cruzi DNA detected by PCR and male patients were at higher risk of progression. These results highlight the importance of T cruzi in the pathophysiology of chronic cardiomyopathy.