Espinosa‑Pereiro et al. Parasites Vectors https://doi.org/10.1186/s13071-019-3518-4 (2019) 12:260 Parasites & Vectors RESEARCH Open Access A retrospective study on the influence of siblings’ relatedness in Bolivian patients with chronic Chagas disease Juan Espinosa‑Pereiro1, Adrián Sánchez‑Montalvá1,2*, Fernando Salvador1,2, Augusto Sao‑Avilés1,3, Elena Sulleiro4 and Israel Molina1,2 Abstract Background: Chagas disease is a protozoan infection caused by Trypanosoma cruzi. The disease has a chronic course in which 20–30% of the patients would develop progressive damage to the cardiovascular system and the gastro‑ intestinal tube. We are still unable to predict who will develop end-organ damage but there are some acquired and genetic risk factors already known. Results: We reviewed data from 833 patients with serologically confirmed Chagas disease in this retrospective study. Patients were classified as siblings or non-siblings (controls) and the results of pre-treatment blood PCR assay, end-organ damage (cardiac and/or gastrointestinal), and the presence of delayed type hypersensitivity (DTH) skin involvement in patients treated with benznidazole were analyzed. Siblings were grouped by family and we randomly generated groups of 2 or 3 persons with the remaining controls. We classified the results of each variable as con‑ cordant or discordant and compared the concordance in these results among the sibling groups with that among control groups. We identified 71 groups of siblings and randomly generated 299 groups of non-related patients. Pre-treatment blood PCR concordance was significantly higher (19%) among siblings compared to controls (P = 0.02), probably due to a higher frequency in pre-treatment positive results. No other statistically significant differences were found. Conclusions: A significant difference was found in the concordance of pre-treatment blood PCR for T. cruzi among siblings compared to non-related controls. Keywords: Chagas disease, Siblings, Brotherhood, Cardiomyopathy, Gastrointestinal disease, Delayed type hypersensitivity Background through infected food). After the initial, acute phase, Chagas disease is caused by the protozoan hemoflagellate Chagas disease has a chronic course in which most T. cruzi and is estimated to affect more than 6–13 mil- patients remain asymptomatic for life, the so-called inde- lion people worldwide. Socioeconomic development and terminate phase of the disease. Over years to decades, migrant flows have caused the disease to change from a about 20–30% of these patients progress to the determi- rural vector-borne disease endemic to Latin America to nate phase of the disease, characterized by cardiac and/or a global disease with different mechanisms of transmis- gastrointestinal manifestations [1, 2]. sion (mother-to-child transmission, tissue and organ In chronic Chagas disease, there is a consensus about transplantation, blood transfusion, and oral transmission the recommendation of treating patients younger than 18 years-old, women at childbearing age, and people *Correspondence: adrian.sanchez.montalva@gmail.com at risk or actually suffering a reactivation of the disease 2 Vall d’Hebron Resarch Institute, University Hospital Vall d’Hebron, (i.e. when receiving immunosuppressant drugs), on the Barcelona, Spain basis of different studies [3–5]. The BENEFIT study has Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Espinosa‑Pereiro et al. Parasites Vectors (2019) 12:260 Page 2 of 8 recently showed no improvement when there is estab- this genetic background could make disease progression lished chronic chagasic cardiomyopathy [6]. The corner- and toxicity outcomes more similar among siblings than stone of the treatment against T. cruzi is benznidazole. among non-related patients and conducted a retrospec- The main drawback of benznidazole is its toxicity, which tive review to compare these variables between both appears in 40–70% of the patients leading to discontinua- groups. tion of treatment in 12–18% of them [7–9]. There is increasing evidence on predisposing fac- Results tors to organ damage and to treatment-related toxic- From the 833 patients with a diagnosis of Chagas disease ity in patients with chronic Chagas disease [10–12]. in our database, we identified 153 siblings in 71 groups. Some of this evidence shows a central role of the host’s With the remainder of the database, 299 groups of non- genetic background in regulating the immune response siblings were randomly generated. The demographic against the parasite, which leads to tissue damage when and clinical characteristics of the full database and the it is improperly regulated [13]. We hypothesized that sibling groups are shown in Table 1. Ninety-five percent Table 1 Demographic characteristics of the siblings grouped in 71 phratries, the remaining controls and the full cohort Variable Siblings (n = 53) (71 Control (n = 685) (299 P-value siblings vs Cohort (n = 833) (%) P-value groups) (%) groups) (%) controls siblings vs cohort Country of origin Argentina 3 (1.96) 15 (2.18) 0.865 18 (2.16) 0.875 Bolivia 144 (94.11) 640 (93.43) 0.757 784 (94.11) 1.000 Brazil 0 2 (0.29) 0.505 2 (0.24) 0.544 Chile 0 1 (0.14) 0.141 1 (0.12) 0.668 Colombia 0 2 (0.29) 0.505 2 (0.24) 0.544 Ecuador 1 (0.65) 4 (0.58) 0.919 5 (0.60) 0.942 El Salvador 0 2 (0.29) 0.505 2 (0.24) 0.544 Honduras 3 (1.96) 5 (0.72) 0.152 8 (0.90) 0.240 Paraguay 0 6 (0.87) 0.247 6 (0.70) 0.299 Spain 2 (1.30) 1 (0.14) 0.028* 3 (0.36) 0.132 Venezuela 0 2 (0.29) 0.505 2 (0.24) 0.544 Sex Female 104 (67.97) 457 (66.71) 0.765 561 (67.34) 0.878 Male 49 (32.02) 223 (32.55) 0.899 272 (32.65) 0.878 Mean age (years) 42 44 44 Pre-treatment PCR 112 (73.20) 495 (73.26) 0.988 607 (72.86) 0.931 Positive 45 (40.17) 200 (29.19) 0.008* 245 (29.41) 0.008* Kushnir 142 (92.81) 637 (93.00) 0.934 779 (93.51) 0.749 K0 119/142 (83.80) 539/637 (84.61) 0.803 658/779 (84.44) 0.841 K1 18/142 (12.67) 77/637 (12.08) 0.840 95/779 (12.19) 0.868 K2 2/142 (1.40) 12/637 (1.88) 0.686 14/779 (1.79) 0.734 K3 3/142 (2.11) 9/637 (1.41) 0.525 12/779 (1.54) 0.609 Barium enema 128 (83.66) 553 (80.72) 0.399 681 (81.75) 0.572 Pathologic 24 (18.75) 119 (17.37) 0.685 143 (21.00) 0.527 Esophagogram 109 (71.24) 421 (61.45) 0.023* 530 (63.62) 0.069 Pathologic 6/109 (5.55) 26/421 (3.79) 0.322 32/530 (6.03) 0.818 Treatment 100 (65.35) 441 (64.37) 0.819 541 (64.94) 0.922 Benznidazole 91/100 (91) 409/441 (93) 0.392 500/541 (92.00) 0.678 Posaconazole 9/100 (9) 32/441 (7.2) 0.445 41/541 (7.57) 0.544 Rash (benznidazole) 16/91 (17.58) 88/409 (21.51) 0.279 104/500 (20.80) 0.3628 *P < 0.05 (statistically significant differences) Abbreviations: n, number of patients; PCR, polymerase chain reaction Espinosa‑Pereiro et al. Parasites Vectors (2019) 12:260 Page 3 of 8 of the patients were born in Bolivia. There was one case result of the esophagogram and barium enema, and the in which one brother was born in Bolivia and the other presence of DTH after benznidazole use). in Argentina. In another group, both brother and sister In the studied data, there was a near 2:1 ratio of female were born in Spain. Sixty-eight percent of the patients to male patients, which may reflect patterns of migra- were female. Pre-treatment PCR assay was performed tion and approach to healthcare, probably under-repre- in a similar proportion in all the groups, but it was posi- senting male involvement in the endemic countries. The tive in a higher proportion among the sibling population vast majority of patients were Bolivian, so the results are than in the general and control population (40 vs 29%, applicable mainly to this population. There was a signifi- Z = 2.64 and Z = 2.65, respectively; both P = 0.008). The cant difference in the country of origin only in the case of proportion of patients to whom an esophagogram was Spanish patients, which represented documented cases performed was different (71% in the sibling group vs of vertical transmission. They were so small in number 61% in the control (Z = 2.27, P = 0.02) and 63% (Z = 1.81, that the likely effect on the global analysis is negligible (2 P = 0.06) in the full database, although the rate of patho- in the sibling groups and 1 in the controls, P = 0.02). logical esophagograms was similar in all groups (4–6%, without statistical significance). PCR The results of the concordance analysis of the valid data The main and only significant difference was found in are summarized in Table 2. Incomplete data from one or the concordance of pre-treatment PCR. Nevertheless, more of the members of a given group led to the loss of a the significantly higher positive result in pre-treatment notable number of groups in the analysis of each variable. blood PCR test among the patients included in the sib- We found a significant difference in the proportion of ling groups as compared to the controls (40% vs 29%, concordant results of the pre-treatment PCR assay (69% P = 0.008) may have biased the results; it must be noted of the sibling groups vs 50% of the controls; χ2 = 4.73, that when splitting the concordant groups on the basis df = 1, P = 0.02), and this difference was still significant of positive or negative results, the biggest difference was after splitting the concordant groups into “all positive” seen between the “all positive” sibling and control groups, and “all negative” of both siblings and controls, due to the with a narrower difference between the “all negatives”. difference in the “all positive”. Results of barium enema Taking into account these limitations, the difference in were more concordant among the siblings than among the concordance of PCR results between siblings points the controls, but the difference did not reach statistical to the presence of genetically determined mechanisms significance (75 vs 62%, χ2 = 2.90, df = 1, P = 0.08), with involved in the relationship between the host and infect- similar rates of available data in both groups (67.60 vs ing T. cruzi. There is growing evidence for the influence 73.91%, χ2 = 1.15, df = 1, P = 0.28). The difference in con- of genetic elements on parasite control by the immune cordance rates of DTH events related to benznidazole system in patients with chronic Chagas disease, but these was very small between siblings and controls (62.5 vs studies are focused on end-organ damage rather than 60.15%, χ2 = 0.06, df = 1, P = 0.80). In the analysis of the on the detection of the relationship of PCR assays and positive vs negative concordance in DTH, a substantial persistent parasitaemia because of the limitations of the difference was found both in siblings and controls with technique. It is known that parasitaemia oscillates dur- more “all negative” groups among siblings than among ing the natural history of the disease, and a positive blood controls (15.62 vs 3.91%, P = 0.04, OR = 3.95, 95% CI: PCR result only indicates that in the moment of the 0.85–18.33), but none of these comparisons reached sampling, there was parasitic DNA in the bloodstream. statistical significance. Cardiovascular evaluation was Furthermore, some acquired conditions are known to similar in both groups, mostly with negative results, modify the results of PCR assays in patients with Chagas resulting in high concordance rates (76.67% in the sib- disease, such as co-infection with helminths, infection lings and 73.4% in the controls, χ2 = 0.27, df = 1, P = 0.59). with human immunodeficiency virus, immunosuppres- When considering esophagogram results the rates of sion and pregnancy [14–17]. In the last 15 years a strong concordant results were similar (89.47 vs 92%, P = 0.74, effort has been made in validation and standardization OR = 0.73, 95% CI: 0.19–3.41), despite a difference in the of these techniques, reaching a sensitivity of 60–90% available data from relatives and non-relatives (53.52 vs for the best performing methods, with a specificity of 42.14%, respectively, χ2 = 3.01, df = 1, P = 0.08). 100% [18, 19]. In our study, pre-treatment PCR was per- formed in 73% of our patients. It was positive in 29.41% Discussion of the patients, the expected sensibility using available In our retrospective study we found a statistical differ- assays in a clinical setting. The difference between the ence in the rate of concordance of pre-treatment PCR, best performing methods and the results obtained in our but not in the remaining variables (Kuschnir status, cohort can be explained by the different sample volume, Espinosa‑Pereiro et al. Parasites Vectors Table 2 Results from the two study groups (2019) 12:260 Variable Siblings groups (n = 71) Control groups (n = 299) P-value Available (%) Concordant (%) Positive (%) Negative (%) Available (%) Concordant (%) Positive (%) Negative (%) Conc Pos/Neg Pre-treatment PCR 39 (54.92) 27 (69.23) 12 (30.77) 15 (38.46) 172 (57.52) 86 (50.00) 23 (13.37) 63 (36.63) 0.023 0.015 Kushnir (>1) 60 (84.50) 46 (76.67) 3 (5.00) 43 (71.67) 297 (99.33) 218 (73.4) 6 (2.02) 212 (71.38) 0.599 0.331 Esophagogram 38 (53.52) 34 (89.47) – 34 (89.00) 126 (42.14) 116 (92.06) – 116 (92.06) 0.740 0.740 Barium enema 48 (67.60) 36 (75.00) 3 (6.25) 33 (68.75) 221 (73.91) 137 (61.99) 8 (3.62) 129 (58.37) 0.088 0.160 DTH 32 (45.07) 20 (62.50) 15 (46.88) 5 (15.62) 128 (42.80) 77 (60.15) 72 (56.25) 5 (3.91) 0.808 0.064 Notes: The first column of each group reflects the availability of data, meaning that data were complete for the two or three members of each phratry or randomly-generated control group. When data of one or more members of the same group were missing, that group was discarded for statistical analysis. The P-values are recorded in the last two columns. The first column (Conc) is the result of comparing concordance among siblings against that among control groups. The second column (Pos/Neg) is the result of comparing the same results differentiating those with concordant positive or negative results Abbreviations: n, number of groups Page 4 of 8 Espinosa‑Pereiro et al. Parasites Vectors (2019) 12:260 Page 5 of 8 conditions of conservation of the sample, the methods Although small, the difference in the results of the used to isolate DNA, the sequences and the reagents concordance of the colonic and the cardiac involvement used, and the thermo-cycling conditions [18]. in our study may reflect different tissue tropism of T. cruzi discrete typing units (DTU) and/or different dam- age pathways for the digestive and the cardiologic form. Cardiomyopathy On one hand, megaesophagus and megacolon are seen We did not find a significant relationship between kin- mainly in patients from the south cone of America and ship and the presence of chagasic cardiomyopathy. The they have been related to specific pathogenicity of the immune response regulation against the parasites infest- DTU present in this area [29]. On the other hand, the ing the myocardium seems to be of central importance in pattern of the inflammatory infiltrates in the gastroin- the development of tissue damage. In patients with cardi- testinal tissue differ from those described in the cardiac omyopathy, the upregulation of this response leads to the form of chronic Chagas disease (inverse CD4/CD8 ratio, deployment of a high-grade, harmful inflammation [20, diminution of CD3+ and CD19+ cells) [30]. Indeed, 21]. Familial aggregation of chagasic cardiomyopathy in the non-classical major histocompatibility class I chain- a cohort of patients with serologically confirmed Chagas related genes A and B (MICA and MICB) seem to rule disease including 247 patients with cardiomyopathy and the interaction of Tγδ, Tαβ CD8+ and natural killer cells 345 controls suggests a genetic component in the regula- with T. cruzi infesting the neuronal and muscular layers tion of this immune response [22]. Different HLA class I of hollow viscera [31]. The association of MICA*011 and and II polymorphisms have been identified as protective HLA-B*14 and DRB1*01 could relate to megacolon [32]. or predisposing factors to the development of chagasic The low rates of end-organ damage detected among cardiomyopathy in different Latin-American popula- the patients in our cohort may mask many differences tions (i.e. a higher frequency of chagasic cardiomyopathy between siblings and controls. These low rates may be with DRB1∗01, DRB1∗08, and DQB1∗0501 in Venezue- explained because the vast majority of our diagnoses are lan patients, and with HLA-DQ1 in Brazilian patients). screening tests of healthy individuals with a mean age of Mutations of the killer cell immunoglobulin-like recep- 44 years old, and with female predominance, so they lack tor genes, TNF-α and caspase pathways have also been the classical risk factors for the development of the deter- related to cardiac involvement [23–27]. minate phase of chronic Chagas disease [33]. As cardiovascular disease (sudden death, end-stage cardiac failure and thromboembolism) is the leading cause of death in patients with Chagas disease, a cardiac DTH screening should be performed in all patients [11, 28]. Our study does not show a higher concordance in DTH Despite that aim, in our cohort the missing evaluations among siblings. Within those treated with benznidazole, invalidated 15.5% of the sibling groups (only 0.67% of the DTH was registered in 17% of the relatives and in the control groups). It is also relevant that cardiac abnormali- 20% of the non-relatives controls. In previous prospective ties have a low prevalence in our cohort (121 patients, studies and clinical trials DTH appeared in a 25–50% of 15.56%, are rated in a Kushnir stage other than 0), and the patients [6–9, 34]. This difference may be explained notably most concordant groups in both siblings and by the incomplete report of mild to moderate adverse controls are due to “all negative” concordance thus mak- events, and the large number of patients lost in the fol- ing unapparent the familiar aggregation of cardiomyopa- low up or who have not completed treatment because thy reported in the aforementioned studies. of adverse events different from skin reactions, or rea- sons not related to toxicity in our retrospective cohort. Indeed, the incidence found in this study is similar to that Gastrointestinal involvement reported of serious adverse events leading to treatment When considering barium enema, concordance was interruption, which are more likely to prompt medi- higher among the siblings than among the controls, but cal assistance and therefore to be registered in clinical the difference did not reach statistical significance (75 vs records. 62%, P = 0.08). As in the case of the cardiac evaluation, Cutaneous DTH has been related to a delayed hyper- there are few “all positive” groups. Regarding the esoph- sensitivity with a Th2 profile (with higher levels of IL-5, agogram, the rates of concordant results were similar IL-10 and eosinophils). A previous study suggested (89.47 vs 92.00%), despite a difference in the available an association between the presence of HLA-B*3505 data from relatives and non-relatives (71 vs 61%). These and moderate-to-severe DTH. This variant is relatively findings may be explained because most studies were frequent among the Bolivian population [10]. Inter- normal in both groups (indeed, there are no “all positive” estingly, although not significant, when splitting con- groups). cordant groups into “all positives” and “all negatives”, a Espinosa‑Pereiro et al. Parasites Vectors (2019) 12:260 Page 6 of 8 bigger proportion of all negatives was found among sib- included in the International Health Program of Catalo- ling groups (15 vs 4%). This finding may reflect a lower nian Health Institute (Programa de Salut Internacional prevalence of HLA-B*3505 and other genetically deter- de l’Insitutt Catalá de Salut, PROSICS), in Barcelona, mined toxicity risk factors. Spain (see Additional file 1: Table S1). It must be noted that the results of the aforementioned Data of 833 patients on the results of blood PCR for T. studies about the genetic determinants of end-organ cruzi before treatment inception, the presence of organic damage in chronic Chagas disease have highly heteroge- damage (cardiac and/or gastrointestinal), and the pres- neous results, even between cohorts of patients from the ence of DTH in patients treated with benznidazole were same country. This is probably due to small sample sizes, reviewed. We identified groups of siblings and used the lack of standardization in study methods (laboratory and remaining patients of the database as controls, randomly statistical), ethnical variations and parasite variability [25, generating non-related patient groups of 2 or 3 individu- 35–41]. The overall immune response against persistent als in a ratio of 4:1 compared to groups of siblings. After T. cruzi infection is likely related to polygenic mecha- the composition of these groups, we anonymized the data nisms, as it depends on a complex cell and cytokine prior to analysis. We analyzed whether the variants were crosstalk [13, 24, 42–44], making it difficult to obtain concordant or not in sibling groups and in the randomly- valuable conclusions from the analysis of the relationship generated non-sibling groups. between a single genetic locus and a disease phenotype. Certain limitations must be considered to contextualize the results of our study. First, as 94.11% of our patients Clinical assessment came from Bolivia, our cohort had a highly homogene- Chagas disease was diagnosed by means of two serologic ous population, which may have masked the influence tests: one based on recombinant antigen (Bioelisa Cha- of familiar genetic background on clinical outcomes gas, Biokit, Lliçà d’Amunt, Spain) and the other based on between siblings. Thus, our results may not be applicable crude antigen (Ortho T. cruzi ELISA, Johnson & Johnson, to patients from underrepresented geographical origins. High Wycombe, UK) [46]. A polymerase chain-reaction Similarly, as the origin of our patients was geographi- (PCR) to detect T cruzi DNA in peripheral blood was cally narrow, they may have been infected with DTU with performed in most patients according to the method similar tissue tropism. Unfortunately, information about described by Piron et al. [47]. strain identification was not available in our cohort as it At the time of the diagnosis, patients were evalu- is not a part of our usual practice. Secondly, ambiental ated with a physical examination, an electrocardiogram factors and re-infections are clear modifiers of the natural (ECG), a chest x-ray and, if there was an abnormal result, history of chronic Chagas disease [45]. In our cohort, re- cardiac ultrasound. Patients were classified according to infections had been interrupted due to an improvement the Kuschnir classification [48]. Gastrointestinal assess- in vector control strategies in the countries of origin and ment was based in symptoms present at initial evalua- to migration to an area where vector-borne transmission tion and barium radiographic imaging of esophagus and had not yet been reported. This could have induced a colon according to Rezende and Ximene’s classifications. slower progression of organ damage, thus masking differ- Patients younger than 55 years were offered treatment ent outcomes in this population. with benznidazole. It must be noted that some patients were treated with posaconazole during a previous study Conclusions [49]. Medication-related adverse events were registered We found a significantly higher proportion of concord- prospectively. For statistical analysis, only cutaneous ance in the result of pre-treatment PCR among Bolivian toxicity was considered as there is some evidence about siblings. A non-significant trend was noted in barium genetic factors influencing this adverse event. enema results and the absence of DTH related to benzni- dazole. Prospective studies with bigger sample sizes may be necessary to identify significant differences in end- Statistical analysis organ involvement of chronic Chagas disease. In each sibling and non-related randomly generated group we evaluated the result of the PCR (positive or Methods negative), Kushnir (grade 0 vs 1 or higher), esophago- Study design and availability of data gram (pathologic or normal), barium enema (patho- We performed a single-center retrospective study based logic or normal) and the presence of DTH within its on the database of patients who received the diagno- members. The possible results were concordant, dis- sis of Chagas disease between 2002 and 2015 at the Vall cordant or missing when data of one or more of the dʼHebron University Hospital, a tertiary care center members of the group were not available. Those groups Espinosa‑Pereiro et al. Parasites Vectors (2019) 12:260 Page 7 of 8 in which data of the considered variable were missing Received: 14 November 2018 Accepted: 20 May 2019 in one or more of its members were excluded for statis- tical analysis. Then we compared the difference in the proportions of concordance between the siblings and the control group using Fisher’s exact test and Pear- References 1. Pérez-Molina JA, Molina I. Chagas disease. Lancet. 2017;391:82–94. son’s Chi-square test. We repeated the test splitting the 2. Carlos J, Dias P. História natural da doença de Chagas. Arq Bras Cardiol. concordant groups into “all positive” and “all negative”. 1995;65:359–66. All data were analyzed with Stata v.13.1 software and R 3. 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