Three hundred and fifty-one multitransfused patients were recruited from three health public institutions located in the city of Campinas, Sao Paulo state, Brazil. The metropolitan region of Campinas, with 3.2 million inhabitants, is located in a non-endemic area of CD, in the most densely populated region of Brazil. Informed consent was obtained from all patients or legal guardian, and the study was approved by the local Institution Review Board. Patients were considered multitransfused if they had been transfused with at least 10 units of blood products or blood derivatives divided between at least two occasions, with the last occasion being at least 15 days apart from recruitment date. One unit of blood product was defined as 1 unit of whole blood, 1 unit of packed red blood cells, 1 unit of a platelet concentrate or platelet apheresis, 1 unit of plasma, 1 unit of cryoprecipitate (Cryo) or 1 vial of lyophilized coagulation factor concentrate. Patients who were aware of a positive test for CD before the first transfusion event were excluded from the study. Clinical and epidemiological data were obtained through a standard interview and from the patient's medical records. This interview was performed by one member of the team using a structured questionnaire after consent was obtained. The questionnaire covered clinical and epidemiological details of the transfusion history of the patients such as: diagnosis, type and number of blood products used, date of first transfusion event and number of facilities in which the patient was transfused. The questionnaire also included questions about alternative routes for transmission of common blood-borne pathogens such as intravenous drug use and previous history of invasive medical interventions and/or alternative medical interventions. In order to exclude other routes of CD transmission in the study population, patients were questioned about living in CD-endemic areas, in dwellings with mud/mud-brick walls or known to be infested with triatomine bugs. Patients were also questioned about relatives or other household contacts with confirmed diagnosis of CD.
Each calendar-year during which the patient received any number of transfusions was recorded as one year of exposure to transfusions. Donor exposure was estimated assuming 1 unit:1 donor for all blood products, except lyophilized factor concentrates that were not included in this estimation, because of the assumption that T. cruzi lose viability during the production of lyophilized clotting factor concentrates. In fact, there are no reports of transmission of CD by these types of blood products.
Blood samples were drawn at the day of recruitment. Serum samples were tested in duplicate using an enzyme immunoassay (Hemobio Chagas, Embrabio, São Paulo, SP, Brazil), according to manufacturer's instructions. The results of anti-T. cruzi test were expressed as the optical densities of the samples (S) divided by the cut-off value (C). S/C values ≤ 0.8 were considered seronegative, S/C values between 0.8 and 1.2 were considered indeterminate and S/C values ≥ 1.2 were considered seropositive. Positive results were confirmed in a second sample, specifically collected for to confirm these results, by immune-fluorescence assay (Imuno Cruzi and Fluoline H, Biolab-Meriéux, RJ, Brazil). For the purpose of this study, samples were considered true positive if they were seropositive in both tests.
The observed prevalence of CD was then calculated. To estimate the impact on the safety of the blood supply of the implementation of 100% coverage of serological screening of CD among blood donors, January 1st 1997 was selected to define the experimental groups. The mean number of units transfused per year of exposure was compared using the Mann-Whitney test and a P < 0.05 was considered significant.