RESEARCH ARTICLE Risk of Trypanosoma cruzi infection among travellers visiting friends and relatives to continental Latin America Adrián Sánchez-Montalvá ID1☯, Catalina Salinas ID2☯, Elena Sullerio3, Fernando Salvador1, Pau Bosch-Nicolau ID1, Clara Crespillo-Andújar4, Elena Trigo ID5, Sı́lvia Roure ID6, Lluı́s Valerio6, Juan Espinosa-Pereiro ID1, Israel Molina ID1* 1 Infectious Diseases Department, Vall d’Hebron University Hospital, Universidad Autónoma de Barcelona, PROSICS Barcelona, Barcelona, Spain, 2 Zoonoses and One Health Masters Program. Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Microbiology Department, Vall d’Hebron University Hospital, a1111111111 PROSICS Barcelona, Barcelona, Spain, 4 National Referral Centre for Tropical Diseases, Infectious Diseases Department, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación a1111111111 Sanitaria, Madrid, Spain, 5 Travel and Tropical Medicine Referral Unit. La Paz-Carlos III University Hospital, a1111111111 Madrid, Spain, 6 North Metropolitan International Health Unit, PROSICS (International Health Program of the a1111111111 Catalan Health Institute), Universitat Autònoma de Barcelona, Barcelona, Spain a1111111111 ☯ These authors contributed equally to this work. * imolina@vhebron.net OPEN ACCESS Abstract Citation: Sánchez-Montalvá A, Salinas C, Sullerio E, Salvador F, Bosch-Nicolau P, Crespillo-Andújar C, et al. (2021) Risk of Trypanosoma cruzi infection Background among travellers visiting friends and relatives to continental Latin America. PLoS Negl Trop Dis Chagas disease (CD) is regarded as a possible risk for travellers to endemic areas of conti- 15(7): e0009528. https://doi.org/10.1371/journal. nental Latin America (LA). The aim of the study is to determine the risk of Trypanosoma pntd.0009528 cruzi (TC) infection among travellers to CD endemic areas and to identify risk factors for Editor: Alberto Novaes Ramos, Jr, Federal acquiring TC infection. University of Ceará, Fortaleza, Brazil, BRAZIL Received: February 18, 2021 Methods/Principal finding Accepted: June 1, 2021 We designed a multicenter cross-sectional study among travellers in Spain (Badalona, Bar- celona and Madrid). All available adults with laboratory confirmed proof of absence of TC Published: July 2, 2021 infection from January 2012 to December 2015 were contacted. Participants referring a trip Copyright: © 2021 Sánchez-Montalvá et al. This is to LA after the negative TC screening were offered to participate. We performed a standard- an open access article distributed under the terms of the Creative Commons Attribution License, ized questionnaire of travel related factors and measurement of TC antibodies in serum. A which permits unrestricted use, distribution, and total of 971 participants with baseline negative TC serology were selected from the microbi- reproduction in any medium, provided the original ology records. After excluding participants not meeting inclusion criteria, eighty participants author and source are credited. were selected. Sixty three (78.8%) were female, and the median age was 38 (IQR 34–47) Data Availability Statement: The data underlying years. The reason to travel was visiting friends and relatives in 98.8% of the participants. the results presented in the study are available under request to the corresponding author, and The median duration of travel was 40 (IQR 30–60) days, with 4911 participants-day of expo- upon approval of the Vall d’Hebron Ethical sure. Seventy seven cases (96.25%) participants had two negative TC serology tests after Committee. The data from this study was approved the travel, two cases (2.5%) had discordant serology results (considered false positive to be used with the aim to fulfill this project, others results) and one case was infected before travelling to LA. According to our data, the upper uses need further ethical approvals. Contact information: Vall d’Hebron Ethical Review Board, limit of the 95% confidence interval of the incidence rate of TC acquisition in travellers is 0.8 Email: ceic@vhir.org. per 1000 participant-days. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009528 July 2, 2021 1 / 11 PLOS NEGLECTED TROPICAL DISEASES Risk of T. cruzi infection in travellers to continental Latin America Funding: ASM was supported by a postdoctoral Conclusions/Significance grant “Juan Rodés” (JE18/00022) from the Instituto de Salud Carlos III (www.isciii.es) through Among 79 non-CD travellers to TC endemic areas, we found no cases of newly acquired TC the Spanish Ministry of economy and infection. The incidence rate of TC acquisition in travellers to endemic countries is less than competitiveness. The funders had no role in study or equal to 0.8 per 1000 traveller-days. design data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Author summary Chagas disease is caused by the protozoan parasite T. cruzi. It is endemic in certain areas of continental Latin America. Few cases of T. cruzi infection have been described in travel- lers. However, there is little information regarding the incidence rate of T. cruzi infection during a trip to continental Latin America. In this study we aim to study the incidence of T. cruzi infection among migrants from Latin America living in Spain travelling to visit friends and relatives. In this study we found no cases of newly acquired T. cruzi infection among 79 previously uninfected travellers and calculate that the upper limit of the 95% CI of the incidence rate of T. cruzi acquisition in travellers is 0.8 per 1000 participants-day. Introduction Concerns that travel to Chagas disease (CD) endemic areas of continental Latin America may pose a risk to acquire CD have been proposed and are part of health travel recommendations. The Yellow Book from Centers for Disease Control and Prevention (CDC) states that “the risk to travellers is extremely low, but they could be at risk if staying in poor-quality housing or from consuming contaminated food or beverages in endemic areas.”[1] Global mobility and travel patterns are rapidly evolving, modifying the dynamic of endemic diseases. CD, once restricted to continental Latin America, is also prevalent in other countries with migrants from this area. Outside Latin America, United State of America and Spain have the largest population with CD. [1] The reason to travel is usually associated with the risk of travel-related diseases. Travellers visiting friends or relatives (VFR) are a group with increased risk of travel- related diseases. Despite there are different definitions for VFR, it usually refers to travellers whose primary purpose of travel is to visit friends or relatives and for whom there is a different epidemiologic risk of acquiring a diseases between the origin and the destination. The great majority of VFR travellers are individuals born abroad returning to their home countries to visit family and friends, although other individuals with other circumstances are also included. [1] In 2010, the European Travel Network reported 60 cases of chronic CD among 7408 returning travellers across Europe.[2] It is possible that these cases of chronic CD reported among VFR travellers actually represent chronic infections not detected prior to travel since pre-travel CD serologies is not routinely performed. The causal agent of CD, or American trypanosomiasis, is the protozoan parasite Trypano- soma cruzi. CD is endemic in 21 countries in continental Latin America and is most com- monly transmitted through triatomine vectors, which are obligate blood-feeder insects. Other routes of transmission are also possible including mother-to-child transmission, oral transmis- sion through beverages infected with the T. cruzi and through blood transfusion and organ transplantation. T. cruzi life cycle encompasses a complex interaction between the wild and domestic environment, where multiple actors are involved. Human activities in enzootic areas modify the behaviour of the vector and wild animal increasing the likelihood of human-vector interaction.[3] Triatomine infestation of human dwellings is facilitated by certain construction PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009528 July 2, 2021 2 / 11 PLOS NEGLECTED TROPICAL DISEASES Risk of T. cruzi infection in travellers to continental Latin America materials: cracks in mud or concrete walls, junctions between adobe bricks, wooden or cane walls, roofs made of palm trees, and earthen floors have been implicated.[4] Having poultry indoors has also been associated with the presence of triatomine in human dwellings.[5] Fully stuccoed-walls have been identified as a protective factor for both human dwellings and animal enclosures.[6] As vector control and surveillance system improve other transmission routes have become more evident. Oral transmission of T. cruzi may lead to acute cardiac alterations from massive parasitemia.[6] Isolated cases and outbreaks have been reported in Argentina, Brazil, Colom- bia, and Ecuador.[7–9] Reported food vehicles of T. cruzi infection include sugar cane, açaı́ juice, palm wine, and wild game.[10] The interaction between humans and triatomine vectors has determined transmission in endemic areas, but globalization is now determining epidemi- ology trends throughout the world. Transmission related to blood products, donation of organs, and mother-to-child occurs worldwide with serious public health implications. [10] For non-endemic countries, determin- ing the risk of acquiring Chagas disease after travel to endemic areas would contribute the evi- dence necessary to support pre-travel advice at the clinical level, and screening efforts at the policy-maker level. Risk of acquiring CD has been deemed to be minimal predicated on cases reported, although risk estimation in low and high risk travellers is lacking. The main objec- tives of this study were 1) to determine the frequency of T. cruzi infection acquired during the travel among returning VFR travellers to T. cruzi endemic areas, 2) to describe VFR travellers demographic and travel characteristics, and 3) to identify possible risk factors for acquiring T. cruzi infection. Methods Ethics statement The Vall d’Hebron institutional review board provided ethical clearance (local review board code number: PR(AG)193/2017). Patients were asked for an oral consent at first telephone contact. Written consent was obtained for all patients that agreed to participate in the study. Study design This is a multicenter cross-sectional study performed from January to December 2017. We recruited all adult subjects with two negative T. cruzi serology tests from January 2012 to December 2015 as provided by the Microbiology Department at 3 Spanish International Health Units: Vall d’Hebron University Hospital (Barcelona, Spain), Hospital La Paz-Carlos III (Madrid, Spain) and Metropolitana Nord Clinic (Badalona, Spain). Travel history was elic- ited by telephone after a brief description of our research and the verbal consent of the patient was granted orally. Eligible subjects were those who travelled to continental Latin America at some point after their latest negative T. cruzi testing. Participants with a recent travel history were requested to wait 2 months before venipuncture. Three telephone calls were made before determining a participant as non reachable. Reachable participants were invited to participate and visit the International Health Unit. Reminders via telephone and text message were sent to ensure attendance. All participants provided a written informed consent, a standardized ques- tionnaire, and a blood sample for T. cruzi serology testing. Exclusion criteria were: previous positive or discordant T. cruzi serology, negative serology after successful treatment for Chagas disease, no history of travel to CD endemic area, participants younger than 18 years and deceased subjects. We analyzed demographic and travel characteristics of participants, determined the fre- quency of newly acquired T. cruzi infection, and identified risk factors of T. cruzi infection PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009528 July 2, 2021 3 / 11 PLOS NEGLECTED TROPICAL DISEASES Risk of T. cruzi infection in travellers to continental Latin America during travel. Consumption of fresh fruit juices and sugar cane was grouped as “beverages”. Accommodation built of adobe, palm tree roofs, walls of cane, or walls of wood was grouped as “house construction materials” for analysis. “Reason for travel” was categorized into tour- ism, business, research or education, missionary or volunteer, and VFR, according to GeoSen- tinel classification.[11] The diagnosis of CD was established by the detection of T. cruzi antibodies using two differ- ent techniques as recommended by the World Health Organization (WHO).[12] Interpreta- tion of the techniques was performed according with the manufacturer’s instructions. Final outcome was considered as follow: positive results to two serologic techniques were considered diagnostic of T. cruzi infection, negative results to two serologic techniques ruled out T. cruzi infection, and one positive and one negative result was considered discordant, and further investigations were recommended. Vall d’Hebron University Hospital and Metropolitana Nord Clinic used two enzyme-linked immunosorbent assays (ELISA), one with recombinant antigen (Bioelisa Chagas, Biokit, Lliçà d’Amunt, Spain) and the other with crude antigen (Ortho T. cruzi ELISA, Johnson and Johnson, High Wycombe, United Kingdom). Hospital La Paz-Carlos III used one ELISA (Vircell Microbiologist, Spain) and one IFI (Indirect Immuno- fluorescence assay; IFA test system, Trinity Biotech, Ireland). A rapid immunochromato- graphic test (ICT; SD BIOLINE Chagas Ab Rapid, Standard Diagnostics, INC, Republic of Korea) that detects the colorimetric antigen-antibody reaction on a membrane is also available. An Internal Board encompassed by 2 clinicians and 2 microbiologists reviewed any positive serology testing for interpretation of the final outcome. Statistical analysis The sample size was a convenience sample, determined by all available eligible subjects between 2012 and 2015, and their willingness to participate. Data were analyzed with STATA (v.14) software. Median and interquartile range (IQR) were calculated for quantitative vari- ables, and frequencies and percentages were calculated for qualitative variables. A regression model was planned to identify risk factors among those with T. cruzi infection and self-report- ing triatomine bugs observation. Variables with a p-value�0.001 in the univariate analysis were included in the multivariable logistic regression model. The analysis to identify risk fac- tors among infected participants could not be performed as none of the participant developed the infection during the study period. Incidence rate of T. cruzi acquisition was calculated as the number new cases divided by total participant-days. 95% upper limit of incidence rate was calculated based on Poisson distribution using Epidat software version 3.1. Results Of the 971 subjects with baseline negative T. cruzi serology between January 2012 and Decem- ber 2015, 169 (17.4%) participants were eligible for study participation. Eighty participants vis- ited the International Health Units and were included in the study and analysis. See Fig 1 for patient flow diagram. Median age was 38 years (IQR 34–47) and 63 (78.8%) were female. The reason to travel was VFR in 98.8% of the participants. The median duration of travel was 40 days (IQR 30–60). Country of birth and country of travel were the same in all but 2 participants: one patient from Bolivia visited relatives in Argentina, and one patient from Colombia visited Peru. Ninety per- cent of the participants travelled for at least 3 weeks. Rural areas were visited by 63.8% of the participants and more than 50% were in contact with farm animal, mainly poultry, swine and cattle. Given the exploratory nature of this study, the participants were interviewed with open PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009528 July 2, 2021 4 / 11 PLOS NEGLECTED TROPICAL DISEASES Risk of T. cruzi infection in travellers to continental Latin America Fig 1. Participants flow diagram. � Participants were untreaceable due to: 308 no one answered the telephone, 86 telephone number did not exist, 26 wrong telephone number, 7 restricted in-coming calls, 20 had no telephone number in the medical record, 15 returned to home countries. �� Participants meeting exclusion criteria: 3 had discordant serology tests, 5 had seroreversion after Chagas’ disease treatment, 22 were deceased and 28 were younger than age 18. https://doi.org/10.1371/journal.pntd.0009528.g001 questions like exposure to peridomestic farm animals during travel. More data are shown in Table 1. Table 2 shows exposure to risk factors for T. cruzi infection. A total of 71.3% of the partici- pants consumed any beverages associated with CD oral infection. Regarding the building materials of accommodation during travel, 17 (21.3%) participants reported staying at a house built of adobe; 3 (3.8%) reported palm tree roof; 4 (5%) reported wooden walls; and 2 (2.5%) reported cane walls. Accommodation built by any of these materials was reported by 18 (22.5%) of participants. Self-reported insect bites were referred by 55% of the participants; none of them were attributed to triatomine bugs. Surprisingly, 66.3% of the participants reported on the observation of triatomine bugs. No participant referred blood transfusion, hospitalization or surgical procedures during the trip. Risk factors associated with self-reporting triatomine bug observation in VFR returning travellers in the univariate analysis were returning from Bolivia, consumption of implicated beverages and having acquaintances with CD. After the multivariable analysis, only the con- sumption of beverages remained statistically significant. More information is shown in Table 3. Among our group of returning VFR travellers to endemic areas, 79 out of 80 (98.75%) had a non positive T. cruzi serology after travel, being negative in 77 participants (96.25%) and dis- cordant inconclusive serology (probably false positive results) in 2 participants. One patient was considered to have had T. cruzi infection before the trip according to the trace-back efforts that included medical records and blood transfusion infection diseases screening records. Table 4 shows the trace-back investigation of the discordant and positive case, along with the internal board final decision. Therefore, according to our data, the upper limit of the 95% con- fidence interval of the incidence rate of T. cruzi acquisition in travellers is 0.8 per 1000 partici- pant-days. Discussion Our study assesses the risk of acquiring CD after travelling to continental Latin America exclu- sively among participants documented to be free of T. cruzi infection before travel. Other PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009528 July 2, 2021 5 / 11 PLOS NEGLECTED TROPICAL DISEASES Risk of T. cruzi infection in travellers to continental Latin America Table 1. Demographic and travel characteristics of study population (n = 80). VSF: visiting friends and relatives. Descriptive data Frequencya %b Demographic characteristics Female sex 63 78.8 Median age (years) 38 IQR 34–47 Travel characteristics Travel destination Bolivia 32 40 Ecuador 14 17.5 Perú 9 11.25 Colombia 7 8.8 Honduras 6 7.5 Brazil 4 5 Argentina 3 3.8 Paraguay 3 3.8 Nicaragua 1 1.3 Venezuela 1 1.3 Reason for travel VFR 79 98.8 Business 1 1.2 Duration of travel (days) 1–30 33 41.3 31–60 28 35 61–90 11 13.8 >90 8 10 Visited rural areas 51 63.8 Peridomestic farm animals 42 52.5 Dogs in or around house 58 72.5 Cats in or around house 42 52.5 Fever during travel 14 17.5 a Absolute frequency for all variables except “Age” which reflects the median value b Percentage for all variables except for “Age” which reflects interquartile range https://doi.org/10.1371/journal.pntd.0009528.t001 Table 2. Exposure to well described risk factors for T. cruzi infection (n = 80). Risk factors described in the literature Frequency % Beveragesa 57 71.3 Fresh fruit juices only 26 32.5 Sugar cane only 4 5 Both 27 33.8 House construction materialsb 18 22.5 Self-reported any insect bite 44 55 Observation of Reduviid bugs 53 66.3 Self-reported Reduviid bug-bite 0 0 Blood transfusion 0 0 a Refers to exposure to implicated beverages (fresh fruit juices and/or sugar cane). b Refers to exposure to accommodation built of adobe, roof made of palm trees, walls of wood, and/or walls of cane. https://doi.org/10.1371/journal.pntd.0009528.t002 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009528 July 2, 2021 6 / 11 PLOS NEGLECTED TROPICAL DISEASES Risk of T. cruzi infection in travellers to continental Latin America Table 3. Factors associated with self-reporting triatomine bugs observation. Triatomine bugs sighting No triatomine bug sighting p value, univariate OR (95% CI), univarate Adjusted OR (95% (n = 53) (n = 27) analysis analysis CI) Sex, female 40 (75.5%) 23 (85.2%) 0.31 0.5 (0.2–1.8) Age, <35 years 8 (29.6%) 22 (41.5%) 0.3 0.6 (0.2–17) Visiting rural areas 34 (64.2%) 17 (63%) 0.92 1.1 (0.4–2.9) Destination, Bolivia 28 (52.8%) 5 (18.5%) 0.003 4.8 (1.6–14.5) 2.1 (0.6–8.2) Contact with farm animals 28 (52.8%) 14 (51.9%) 0.93 1.1 (0.4–28) Contact with dogs 41 (77.4%) 17 (63%) 0.17 2 (0.7–5) Contact with cats 32 (60.4%) 10 (37%) 0.048 2.7 (1–7.1) Consumption of implicated 43 (81.1%) 14 (51.9%) 0.006 4.4 (1.6–12.6) 3.3 (1.1–9.8) beverages Relative or friend with CD 28 (52.8%) 4 (14.8%) 0.001 6.7 (2.0–22.1) 3.8 (0.9–15.8) House construction materialsa 13 (24.5%) 5 (18.5%) 0.54 1.5 (0.5–4.7) Note: CD, Chagas disease a Refers to exposure to accommodation built of adobe, roof made of palm trees, walls of wood, and/or walls of cane. https://doi.org/10.1371/journal.pntd.0009528.t003 publications have reported cases of T. cruzi infection among travellers.[2] However, no docu- mentation of T. cruzi infection-status before travel was available, precluding us from assuring that those cases were infected during travel to endemic areas. Our study participants had a baseline T. cruzi testing as part of screening programs of asymptomatic migrants of Latin American origin at the Infectious Diseases Department, routine blood bank screening proto- col, congenital transmission prevention program, diagnostic workup of clinical findings com- patible with CD, prior to immunosuppressant therapy, biologic therapy, or organ transplantation. Among 79 adult VFR travellers to continental Latin America and who were free of CD prior to travel, we found no cases of T. cruzi infection and found 2 patients with dis- cordant serology that were classified as inconclusive and probably false positive results by our Internal Board. In our study, the incidence rate of acquisition of T. cruzi infection in travellers to Latin America CD endemic areas is less than is 0.8 per 1000 participants-day. VFR travellers in this study were migrants from Latin America living in Spain with demo- graphic characteristics similar to Latin American migration to Spain. However, the methodology of the study may have introduced a selection bias, limiting the extrapolation of the data to a broader population. Similar to a previous European studies, a female predominance among Latin American VFR travellers was noted.[] [13,14] The median duration of travel was 40 days (IQR 30–60 days), which is much longer than that found in other series of ill returning travellers (median 17 days, IQR 8–43).[15] Exposure to risk factors for T. cruzi infection described in the literature was assessed with a standardized questionnaire. Fresh fruit juices and sugar cane bever- ages were common practices among our study cohort. These beverages can be contaminated with the T. cruzi infected vector itself or its faeces and be inadvertently ingested. [7–9] Regarding the quality of accommodations, 22.5% of participants stayed at a house built of adobe, palm tree- thatching, wooden walls, or cane walls. Poor dwelling conditions are associated with triatomine bug domestic infestation, increasing the risk of T. cruzi infection acquisition. Despite efforts aimed at vector control have been successfully implemented in Latin America since the 1990s, with a significant reduction of vectorial transmission in many countries and eradication in oth- ers, it seems necessary to maintain such programs and implement a permanent surveillance sys- tem to rapidly detect good dwelling practices deterioration or/and triatomine infestation.[4] We found 3 cases with at least one positive serology test result. After carefully evaluation of the trace back investigation results by the Internal Board, 1 case was considered to be infected PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009528 July 2, 2021 7 / 11 PLOS NEGLECTED TROPICAL DISEASES Risk of T. cruzi infection in travellers to continental Latin America Table 4. Trace back investigation of participant with a positive serology testing and internal board final decision (shade results are before traveling to T. cruzi endemic countries). Case 1 (Bolivia, Cochabamba) Transfusion center Dec 2013 May 2014 Aug 2014 Jan 2015 Jul 2016 Jan 2018 ELISA (native) Inderterminate Negative Negative Negative - - IFI Indeterminate Indeterminate Indeterminate Positive - - ELISA (recombinant) Positive Positive Positive Indeterminate - - PCR peripheral blood - Negative Negative - - - Tertiary Hospital Rapid Test (ICT) - Negative Negative - Negative Negative ELISA - Negative Negative - Negative Indeterminate IFI - - - - - positive PCR peripheral blood - - - - - Negative Internal Board Final Decision Inconclusive Case 2 (Bolivia, Cochabamba) Transfusion center Jul 2016 Jan 2018 Feb 2018 ELISA (native) - - - IFI Positive - - ELISA (recombinant) - - - Chemiluminescence Positive PCR peripheral blood - - - Tertiary Hospital Rapid Test (ICT) Negative Negative Negative ELISA Negative Positive Positive IFI - Positive Positive PCR peripheral blood Negative Negative Negative Internal Board Final Decision Chronic infection previous to travel (2016 Hospital False negative) Case 3 (Argentina, Buenos Aires) Tertiary Hospital Dec 2015 May 2017 Aug 2017 ELISA (recombinant) Negative Positive Positive ELISA (native) Negative Negative Negative PCR peripheral blood - - Negative Internal Board Final Decision Inconclusive ELISA: enzyme-linked immunosorbent assays; ICT: immunochromatographic serological test; IFI: indirect immunofluorescence; PCR: Polymerase chain reaction; https://doi.org/10.1371/journal.pntd.0009528.t004 before the trip and the other 2 cases were deemed as inconclusive (probably false positive results). Discordant serology testing poses a diagnostic challenge for the clinician. A study by Moure et al using a western blot technique (TESA-Blot (Biomérieux, RJ, Brazil)) was evaluated as a confirmatory diagnostic tool in patients with inconclusive and discordant serology helped establish the diagnosis of CD in half of cases, unfortunately the technique was not available in our laboratories for further investigation.[16] These patients will be followed up in clinic with repeated serology every 6 months. Currently, clinical trials of new treatment in CD include travelling to endemic areas as exclusion criteria limiting the number of available participants or withdrawing participants due to unexpected trips.[17,18] According to our results this exclusion criteria should be PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009528 July 2, 2021 8 / 11 PLOS NEGLECTED TROPICAL DISEASES Risk of T. cruzi infection in travellers to continental Latin America reconsidered in future clinical trials, as risk of T. cruzi reinfection is minimal. However, trials developed in areas where vectorial transmission is still present should try to minimize the risk of reinfection by implementing strategies to improve dwelling conditions, to undertake peri- domestic conditioning, to induce behavioural modifications, to work in the relocation of ani- mal facilities and further work into vector control strategies. [19,20] This study has several limitations. First, we selected subjects that had already approached the health care system, and tested negative for CD, so this strategy might have pre-selected sub- jects with a lower predisposition or from areas with lower incidence of CD. However, many of them had relative or friends with CD, which may indicate current or previous active transmis- sion in the visit area. Second, the necessity to reach participants by telephone to assess for eligi- bility significantly reduced our sample size since almost half of the subjects from our database were untraceable after several attempts. This was especially true for the years 2012 and 2013. Presumably these subjects do not keep the same telephone number for a long period of time. Another limitation was that half of eligible subjects were unable to come to the study site due to schedule or distance limitations. Jobs filled by foreign workers often have longer hours and offer little or no time flexibility. Female participants tended to work caring for children or the elderly, which gave them more time flexibility than their male counterparts. Participants may have wanted to “save” work leaves for more urgent needs. Working hours at our study site overlapped with usual working hours, which may have introduced a selection bias to represent more females. Conclusions Among 79 non-CD VFR travellers to continental Latin America, we found no cases of newly T. cruzi infection. Two participants had inconclusive results due to discordant results, likely from false positive results. The incidence rate (risk of acquiring) of T. cruzi acquisition in trav- ellers to endemic countries is less than or equal to 0.8 per 1000 travellers-day. This study may help improve pre-travel health counselling and better inform travellers to continental Latin America of the low risk of T. cruzi infection acquisition. However, mitigating action should still be recommended. Besides, they can also serve to minimize the risk of acquiring other travel-related infections. Author Contributions Conceptualization: Adrián Sánchez-Montalvá, Elena Trigo, Israel Molina. Data curation: Adrián Sánchez-Montalvá, Catalina Salinas, Fernando Salvador, Pau Bosch- Nicolau, Clara Crespillo-Andújar, Elena Trigo, Sı́lvia Roure, Lluı́s Valerio. Formal analysis: Adrián Sánchez-Montalvá, Catalina Salinas, Fernando Salvador, Clara Cre- spillo-Andújar, Sı́lvia Roure, Lluı́s Valerio, Juan Espinosa-Pereiro, Israel Molina. Investigation: Adrián Sánchez-Montalvá, Catalina Salinas, Elena Sullerio, Fernando Salvador, Pau Bosch-Nicolau, Clara Crespillo-Andújar, Elena Trigo, Sı́lvia Roure, Lluı́s Valerio, Juan Espinosa-Pereiro, Israel Molina. Methodology: Adrián Sánchez-Montalvá, Catalina Salinas, Elena Sullerio, Fernando Salvador, Pau Bosch-Nicolau, Clara Crespillo-Andújar, Elena Trigo, Sı́lvia Roure, Lluı́s Valerio, Juan Espinosa-Pereiro, Israel Molina. Project administration: Catalina Salinas. Supervision: Adrián Sánchez-Montalvá, Israel Molina. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009528 July 2, 2021 9 / 11 PLOS NEGLECTED TROPICAL DISEASES Risk of T. cruzi infection in travellers to continental Latin America Validation: Adrián Sánchez-Montalvá, Elena Sullerio, Elena Trigo, Lluı́s Valerio, Israel Molina. Writing – original draft: Adrián Sánchez-Montalvá, Catalina Salinas. Writing – review & editing: Adrián Sánchez-Montalvá, Elena Sullerio, Fernando Salvador, Pau Bosch-Nicolau, Clara Crespillo-Andújar, Elena Trigo, Sı́lvia Roure, Lluı́s Valerio, Juan Espinosa-Pereiro, Israel Molina. References 1. Trypanosomiasis, American (Chagas Disease)—Chapter 4–2020 Yellow Book | Travelers’ Health | CDC. [cited 8 Jan 2020]. Available: https://wwwnc.cdc.gov/travel/yellowbook/2020/travel-related- infectious-diseases/trypanosomiasis-american-chagas-disease 2. 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