Screening of at‐risk blood donors for Chagas disease in non‐endemic countries: Lessons from a 2‐year experience in Tuscany, Italy

Abstract Background Chagas disease (CD) is caused by the protozoan parasite Trypanosoma cruzi and is transmitted by blood‐sucking triatomine insects in endemic areas of Latin America. Transmission can also occur via blood transfusion and is a major cause of CD in non‐endemic areas. Objectives The aim of the study was to assess the prevalence of anti‐T. cruzi antibodies in blood donors at risk of infection in Tuscany, Italy, following the introduction of blood safety Italian legislation. Material and methods Donors (N = 1985) were tested in 2016 to 2018 for anti‐T. cruzi IgG using an immunochromatographic test (ICT). Chemiluminescent immunoassay (CLIA) was performed on ICT‐positive donors to exclude CD, whereas enzyme‐linked immunosorbent assay and western blot were performed in case of discordant results. All assays were performed on CD patients (N = 10) for validation. Results Ten blood donors had a positive ICT result, with a resulting T. cruzi seroprevalence of 0.5% but demonstrated negative results to CLIA, as well as to the other serological assays. The comparison of serological assays suggested a lower relative sensitivity of ICT. Conclusion The results of this study confirm the significance of serological testing in the screening strategy for CD. However, they provide evidence for discontinuing the use of ICT as a screening test and suggest that a sensitive, specific and multi‐sample format assay should be used at the national level for uniformity of results.

transplant of solid organs or haematopoietic stem cells, consumption of food or drinks contaminated by vectors or their faeces and via laboratory accidents. 1 The acute phase of the infection, although mostly asymptomatic, may present with fever, inflammation at the inoculation site (chagoma), unilateral palpebral oedema (Romaña sign), lymphadenopathy and hepatosplenomegaly. After 4-6 weeks, the acute phase resolves spontaneously, and blood parasitaemia reduces substantially, but patients remain chronically infected, and in 30%-40% of them, chronic disease may occur up to 30 years later, with cardiomyopathy and/or megaviscera as the most common clinical manifestations.
In endemic countries, due to multinational control initiatives, the epidemiological situation has dramatically improved, with a significant decline in the incidence of the disease in the last decade. For example, in Argentina, one of the most affected countries in Latin America, a decrease of the infected population has been estimated from 23.0% in 2005 to 5.2% in 2010. 1 In contrast, in non-endemic countries, a dramatic increase in the number of CD cases has been reported in the last decades, changing the scenario of the disease into a worldwide public health concern. 2 For example, in the United States and in Canada, more than 325 000 and between 4000 and 6000 cases of CD have been estimated, respectively. As for Europe, in Spain and Italy, the two countries with the highest number of Latin American immigrants, around 70 000 to 76 000 and 90 000 to 10 000 infected individuals have been estimated, respectively. 1 According to the World Health Organization (WHO), for the diagnosis of CD, at least two serological tests based on different principles should be performed to detect anti-T. cruzi antibodies. 3 Different strategies have been adopted in order to reduce the risk of transfusion-transmitted (TT) CD. Currently, in endemic areas, all donations should be analysed for T. cruzi antibodies. In non-endemic countries, interventions are different: exclusion of at-risk donors (Sweden), T. cruzi serology screening of at-risk blood donors (Portugal, Spain, France and-more recently-Italy) or one-time serological testing of all donors (United States) 4 (Angheben, personal communication). Where serological screening is performed, a single test is considered sufficient to decide on exclusion from donation. 1 For its part, the WHO recommends the use of a single enzyme-linked immunosorbent assay (ELISA) for blood bank screening. 3 The aim of this study was to assess the seroprevalence of T. cruzispecific antibodies in a population of at-risk blood donors from Tuscany, Italy, in a 2-year period following the introduction of the Italian National Blood Centre regulation in November 2015 (L.219, D.M. 02/11/2015). This requires the identification of at-risk candidate donors (those born in Latin America, born from Latin American mother or travellers with history of rural or outdoor activities in endemic areas) through a questionnaire, followed by testing for anti-T. cruzi antibodies with immunological techniques, without any further specification on the characteristics of the test itself. Therefore, samples from both at-risk blood donors and CD patients were tested with different serological methods in order to provide evidence for the amelioration of the current screening strategy. has an in-and out-patient service, and more than 400 individuals have been diagnosed and offered treatment for CD in the last decades.
Samples of blood from 10 patients born in Bolivia who were recruited during screening surveys of Latin American communities between 2012 and 2015 and underwent serology diagnosis for CD through two ELISA assays (details provided below) have been selected as controls for the current study. Nine individuals tested positive to both tests, chronic CD was diagnosed (indeterminate phase), and they were therefore used as positive controls. One patient tested negative for both tests and was therefore considered a negative control.

| Serological methods
Six different assays currently present in the Italian market were performed for the detection of anti-T. cruzi antibodies in serum samples from ICT-seropositive candidate donors, as well as from control patients, to compare the performance of methods.

| Immunochromatographic test
The ICT method Chagas Quick Test (Cypress Diagnostics) was performed according to manufacturer's instructions. The ICT has a strip format, and it is based on T. cruzi recombinant antigen includ-

| Chemiluminescent immunoassay
The chemiluminescent immunoassay (CLIA) method Architect System Chagas (Abbott) was performed following manufacturer's instructions using the instrument Architect i2000 SR (Abbott). The

| Enzyme-linked immunosorbent assay 2
The ELISA method Bioelisa Chagas (Biokit) was performed following manufacturer's instructions. The ELISA has a 96-well plate format,  WHO guidelines for the diagnosis of chronic CD recommend performing a third serological test in case of discordance of the first and second tests. 3    Note: The table shows the results of different serological methods for the detection of T. cruzi antibodies in ICT-positive, at-risk blood donors (N = 10). "…" denotes data not available because of lack of sera. Abbreviations: ELISA1, ELISA using recombinant antigens; ELISA2, ELISA using lysate antigen; WB1, WB using TcF recombinant antigen; WB2, WB using lysate antigen.
positive result for all tests, whereas one patient had a positive result for all tests but ICT (Table 3). These results would suggest a lower sensitivity of ICT compared to other serological methods and therefore provide evidence for the need to replace ICT as the screening test.

| DISCUSSION
When individuals from CD endemic countries migrate to non-endemic countries and act as donors (blood or other cellular products), there is a need to prevent transmission through transfusion or transplantation. 5 In this way, it is possible to guarantee the safety of blood and its products, simultaneously maintaining the blood supply from candidate donors, 6 as shown in the United States where selective T. cruzi screening is nearly equally effective as universal screening, but at a reduced cost. 7 In this study, a low prevalence of seropositive individuals (0.50%) has been observed among blood donors identified to be at risk for ongoing, which showed that the sensitivity of ICT was not optimal (82.8%) in non-endemic countries. 9 It is therefore important to acknowledge the fact that, in light of the non-optimal sensitivity of the ICT assay used for screening, the observed prevalence of seropositive  In addition to donor selection, other strategies may increase transfusion safety, such as pathogen inhibition methods. 10,11 These methods would lower the risk not only of TT CD but also of other parasitic diseases such as malaria, babesiosis and leishmaniosis whose importance in transfusion medicine is often neglected. 12