RESEARCH ARTICLE Impact of Helminth Infection on the Clinical and Microbiological Presentation of Chagas Diseases in Chronically Infected Patients Fernando Salvador1*, Elena Sulleiro2, Adrián Sánchez-Montalvá1, Mónica Martínez- Gallo3, Eugenia Carrillo4, Israel Molina1 1 Department of Infectious Diseases, Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona, PROSICS Barcelona, Barcelona, Spain, 2 Department of Microbiology, Vall d’Hebron University Hospital, PROSICS Barcelona, Barcelona, Spain, 3 Immunology Division, Vall d’Hebron University Hospital, a11111 Barcelona, Spain, 4 WHO Collaborating Centre for Leishmaniasis, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain * fmsalvad@vhebron.net Abstract OPEN ACCESS Citation: Salvador F, Sulleiro E, Sánchez-Montalvá A, Martínez-Gallo M, Carrillo E, Molina I (2016) Background Impact of Helminth Infection on the Clinical and Helminth infections are highly prevalent in tropical and subtropical countries, coexisting in Microbiological Presentation of Chagas Diseases in Chagas disease endemic areas. Helminth infections in humans may modulate the host Chronically Infected Patients. PLoS Negl Trop Dis 10 (4): e0004663. doi:10.1371/journal.pntd.0004663 immune system, changing the Th1/Th2 polarization. This immunological disturbance could modify the immune response to other infections. The aim of this study is to evaluate the rela- Editor: Michael H. Hsieh, George Washington University, UNITED STATES tionship between clinical, microbiological and epidemiological characteristics of Chagas disease patients, with the presence of helminth infection. Received: February 8, 2016 Accepted: April 5, 2016 Methods Published: April 26, 2016 A prospective observational study was conducted at Vall d’Hebron University Hospital (Bar- Copyright: © 2016 Salvador et al. This is an open celona, Spain). Inclusion criteria were: age over 18 years, diagnosis of Chagas disease, access article distributed under the terms of the and not having received specific treatment for Chagas disease previously to the inclusion. Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any The study protocol included Chagas disease assessment (cardiac and digestive evaluation, medium, provided the original author and source are detection of T. cruzi DNA measured by PCR in peripheral blood), and helminth infection credited. diagnosis (detection of IgG anti-Strongyloides stercoralis by ELISA, microscopic examina- Data Availability Statement: All relevant data are tion of stool samples from three different days, and specific faecal culture for S. stercoralis within the paper and its Supporting Information files. larvae). Funding: This research received no specific grant from any funding agency in the public, commercial, or Results not-for-profit sectors. This study was supported by the 6th National Plan (PN) of Research + Overall, 65 patients were included, median age was 38 years, 75.4% were women and Development + Innovation (I+D+I) 2008-2011, ISCIII- most of them came from Bolivia. Cardiac and digestive involvement was present in 18.5% General Division Networks and Cooperative and 27.7% of patients respectively. T. cruzi PCR was positive in 28 (43.1%) patients. Hel- Research Centres + FEDER funds + Collaborative minth infection was diagnosed in 12 (18.5%) patients. No differences were observed in clini- Research Network on Tropical Diseases (RICET): RD12/0018/0020 and RD12/0018/0011. The funders cal and epidemiological characteristics between patients with and without helminth had no role in study design, data collection and infection. Nevertheless, the proportion of patients with positive T. cruzi PCR was higher PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004663 April 26, 2016 1/8 Chagas Disease and Helminth Infection analysis, decision to publish, or preparation of the among patients with helminth infection compared with patients without helminth infection manuscript. (75% vs 35.8%, p = 0.021). Competing Interests: The authors have declared that no competing interests exist. Conclusions We observed a high prevalence of S. stercoralis infection among chronic Chagas disease patients attended in our tropical medicine unit. Strongyloidiasis was associated with signifi- cantly higher proportion of positive T. cruzi RT-PCR determined in peripheral blood. Author Summary Helminth infections (viz. Strongyloides stercoralis, hookworms, Ascaris lumbricoides, Tri- churis trichiura) are highly prevalent in tropical and subtropical areas, and some of these infections may persist in the human host for many years after leaving the endemic area. It is known that helminth infection in humans may modulate the host immune system. This immunological disturbance could modify the immune response to other infections or the antibody production after vaccination. We prospectively studied a group of patients with chronic Chagas disease, with the aim of evaluate the impact of helminth co-infection in the clinical manifestations and microbiological features of Chagas disease. We observed a high prevalence of helminth infection (mostly due to S. stercoralis infection) among chronic Chagas disease patients attended in our tropical medicine unit. Strongyloidiasis was associated with significantly higher proportion of positive T. cruzi RT-PCR deter- mined in peripheral blood. These data increase the scarce available information to under- stand the role of PCR techniques in the management of Chagas disease patients. Further studies are needed to deepen and confirm this interesting relationship. Introduction Chagas disease is a parasitic infection caused by the hemoflagellated protozoan Trypanosoma cruzi. Chagas disease is an endemic disease of Latin America affecting rural and poor popula- tion; nevertheless, progressive urbanization and the increase of population mobility during last decades, have made Chagas disease an urban and global disease outside endemic countries: mainly in United States and Spain [1, 2]. After the acute phase of the infection, a subsequent usually asymptomatic chronic stage (or indeterminate phase) takes place during years; after 20–30 years, up to a 30–40% of patients will develop the symptomatic chronic phase, with cardiac and/or digestive involvement [2]. Chagas disease diagnosis in the chronic phase is based on serological tests. Due to the low para- sitaemia in this phase, classical direct parasitological tests (microhematocrit, hemoculture, xenodiagnose) are usually negative [3]. Nevertheless, more sensitive tests such as the polymer- ase chain reaction (PCR) have being developed [4]. The percentage of positive T. cruzi PCR in peripheral blood in patients with Chagas disease in the chronic phase highly varies depending on the study: it ranges from 80% to 90% in studies performed in endemic countries, and is lower in non-endemic countries, ranging from 28% to 66% [5–10]. T. cruzi PCR is not rou- tinely performed in the management of chronic Chagas disease patients, but is becoming very useful in specific situations, such as follow-up in immunosuppressed patients in order to detect reactivation or in clinical trials to detect treatment failures [11–13]. However the role of the T cruzi PCR in the chronic phase of Chagas disease needs to be defined. PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004663 April 26, 2016 2/8 Chagas Disease and Helminth Infection Helminth infections (viz. Strongyloides stercoralis, Necator americanus, Ancylostoma duode- nale, Ascaris lumbricoides, Trichuris trichiura) are highly prevalent in tropical and subtropical areas, coexisting in Chagas disease endemic areas, and some of these infections may persist in the human host for many years after leaving the endemic area [14]. It is known that helminth infection in humans may modulate the host immune system, changing the Th1/Th2 polariza- tion. This immunological disturbance could modify the immune response to other infections or the antibody production after vaccination [15, 16]. The aim of the present study is to evaluate the relationship between clinical and epidemio- logical characteristics of chronic Chagas disease patients, with the presence of helminth infection. Materials and Methods Ethics statement The study protocol was approved by the Ethical Review Board of the Vall d’Hebron University Hospital (Barcelona, Spain), and written informed consent was obtained from all patients. Pro- cedures were performed in accordance with the ethical standards laid down in the Declaration of Helsinki as revised in 2000. Study protocol This is a prospective observational study performed at the Infectious Diseases Department of the Vall d’Hebron University Hospital, a tertiary hospital included in the International Health Program of the Catalan Health Institute (PROSICS Barcelona, Spain), from March 2014 to February 2015. All adults (over 18 years old) with recently diagnosis of Chagas disease in the chronic or indeterminate form attended during the study period were offered to participate. Exclusion criteria included: previous treatment for Chagas disease or helminth infections, preg- nancy or immunosuppression. Diagnosis of Chagas disease was performed through two positive different serological tests according to WHO recommendations [17]: an enzyme-linked immunosorbent assay (ELISA) with recombinant antigen (Bioelisa Chagas, Biokit, Lliçà d’Amunt, Spain), and an ELISA with crude antigen (Ortho T.cruzi ELISA, Johnson & Johnson, High Wycombe, United Kingdom). Cardiac and digestive involvement was assessed through a clinical symptoms questionnaire, physical examination, 12-lead electrocardiography, chest radiography, and barium enema. Patients were stratified according to the clinical Kuschnir classification for cardiac involvement assessment [18]. Pathologic barium enema was defined by dolichocolon or sigmoid diameter > 6cm (megacolon) [19]. A real time PCR (RT-PCR) to detect T. cruzi DNA in peripheral blood was performed in all patients according to the method described by Piron et al [20]. For helminth infection diagnosis, microscopic examination of stool samples from three dif- ferent days after concentration techniques using Ritchie’s formalin-ether technique were per- formed in all patients. A faecal culture for S. stercoralis larvae detection (charcoal culture) was also performed. Moreover, blood cell count to detect presence of eosinophilia (defined as 500 cells/mm3 and/or 7%), and detection of serum IgG anti-S. stercoralis by ELISA (SciMedx Corporation, Denville, NJ, United States) were conducted. Definition of helminth infection included: confirmed infections through direct observation, and probable infection (presence of eosinophilia and positive S. stercoralis serology in the absence of other causes of eosinophilia). PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004663 April 26, 2016 3/8 Chagas Disease and Helminth Infection Statistical analysis Categorical data are presented as absolute numbers and proportions, and continuous variables are expressed as medians and ranges. The χ2 test or Fisher exact test, when appropriate, was used to compare the distribution of categorical variables, and the Mann-Whitney U test for continuous variables. Results were considered statistically significant if the 2-tailed P value was <0.05. SPSS software for Windows (Version 19.0; SPSS Inc, Chicago, IL, USA) was used for statistical analyses. Results Overall, 72 patients were included during the study period. Six patients were excluded because they did not complete the study protocol; therefore, 66 patients were analyzed. The median age of patients was 38 (18–67) years, and 50 (75.8%) were women. The vast majority came from Bolivia (64 patients, 97%) and, at the time of the first visit, the median duration of residence in our country was 9 (1–14) years, and 41 (62.1%) patients had traveled again to their countries after arriving to Spain (most of them spent less than 2 months in their countries, and stayed in an urban setting). Cardiac involvement was diagnosed in 12 (18.2%) patients (nine patients in the stage I, and three patients in the stage II of the Kushnir classification respectively). Eighteen (27.3%) patients presented abnormalities in the barium enema: 16 patients with dolichocolon, and 2 patients with megacolon. At the time of Chagas disease diagnosis, T. cruzi RT-PCR in peripheral blood was positive in 28 (42.4%) patients. Helminth infection was diagnosed in 12 (18.2%) patients: two patients with confirmed infection (one patient with S. stercoralis and another patient with Hymenolepis nana), and 10 patients with probable infection. Patients with helminth infection had a median eosinophil cell count of 500 (100–1200) cells/mm3. Table 1 shows other protozoan parasites observed in the microscopic examination of stool samples. When comparing main clinical and epidemiological characteristics between patients with and without helminth infection, no differences were observed (Table 2). Nevertheless, the percentage of patients with positive T. cruzi RT-PCR was higher in patients with helminth infection compared with those without helminth infection (75% versus 35.2%, p = 0.021). Discussion We prospectively studied 66 adult patients with Chagas disease to evaluate the relationship between microbiological, clinical and epidemiological characteristics with the presence of Table 1. Protozoan parasites observed in stool samples of Chagas disease patients. Parasites isolated Number of patients (n = 66) Parasites of uncertain significance Blastocystis hominis 23 (34.8%) Dientamoeba fragilis 7 (10.6%) Non pathogenic Entamoeba coli 6 (9.1%) Entamoeba sp 3 (4.5%) Iodamoeba butschlii 3 (4.5%) Endolimax nana 2 (3%) NOTE. Data are reported as number (%) of patients. doi:10.1371/journal.pntd.0004663.t001 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004663 April 26, 2016 4/8 Chagas Disease and Helminth Infection Table 2. Main clinical and epidemiological characteristics of Chagas disease patients visited at Vall d’Hebron University Hospital from March 2014 to February 2015. Overall Patients with helminth infection Patients without helminth infection P (n = 66) (n = 12) (n = 54) value Age, years 38 (18–67) 41 (28–65) 38 (18–67) 0.247 Gender, male 16 (24.2%) 5 (41.7%) 11 (20.4%) 0.145 Time of residence in our country, 9 (1–14) 9.5 (1–14) 9 (1–14) 0.967 years Cardiac involvement 12 (18.2%) 3 (25%) 9 (16.7%) 0.376 Digestive involvement 18 (27.3%) 3 (25%) 15 (27.8%) 1 Positive T. cruzi RT-PCR 28 (42.4%) 9 (75%) 19 (35.2%) 0.021 NOTE. Data are reported as number (%) of patients or median (range). doi:10.1371/journal.pntd.0004663.t002 helminth infection. Positive T. cruzi RT-PCR was more frequent in patients with helminth infection compared with those without helminth infection. Although the study was carried out in a limited group of Chagas disease patients, clinical and epidemiological characteristics found in the study population were similar to those found in larger studies performed in non-endemic countries: most of them coming from Bolivia, young people, majority of women, and low prevalence of cardiac and digestive involvement [2, 8–10]. Therefore, our study population is representative of the Chagas disease population diag- nosed and treated in non-endemic areas. Helminth infection has been diagnosed in 18.2% of the study population, being strongyloi- diasis the most frequent infection (all except from one). The S. stercoralis infection predomi- nance was rather expected, since the median time of residence in Spain in our population was 9 years, thus decreasing the probability of other helminth infections such as Ascaris lumbricoides, hookworms or Trichuris trichiura. S. stercoralis is distributed worldwide, being more frequent in tropical and subtropical areas. High prevalence has been found in Latin American countries where Chagas disease is also endemic, hence co-infection is supposed to be high in this area [21]. Scarce information about the prevalence of strongyloidiasis in Bolivia is available, and it is centered in at risk groups [22]. A study published by Ramos et al showed a S. stercoralis sero- prevalence of 44.4% among Bolivian immigrants living in Spain [23]. Although our study was not focused on intestinal protozoa, it is important to note the high prevalence of Blastocystis hominis and Dientamoeba fragilis infections (34.8% and 10.6% respectively) observed in our study population; despite their pathogenicity remains uncertain and controversial, the presence of these parasites may be used as a marker of potential exposure to other pathogenic parasites. When comparing epidemiological, clinical and microbiological characteristics between patients with and without helminth infection, the first group had statistically significant higher proportion of positive T. cruzi RT-PCR in peripheral blood than the second group (75% and 35.2% respectively). To our knowledge, no previous study has addressed the possible implica- tions of Chagas disease and helminth co-infection in humans. Nevertheless, some interesting studies in animal model have been published with similar findings. Monteiro et al described higher prevalence of T.cruzi-positive blood cultures in golden lion tamarins infected with T. cruzi when they were co-infected with intestinal helminths of the Trichostrongylidae family, which is coherent with the results obtained in our study [24]. Another study performed with T. cruzi infected mice went in depth in this relationship between helminth infection and T. cruzi parasitaemia: no differences in the parasitaemia were found between non co-infected and early co-infected mice (the T. cruzi infection took place 2–4 weeks after Taenia crassiceps infection), PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004663 April 26, 2016 5/8 Chagas Disease and Helminth Infection however, late co-infected mice (the T. cruzi infection took place 8–12 weeks after Taenia crassi- ceps infection, when a predominant Th2-type cytokine response is expected) showed signifi- cantly higher parasitaemia compared with non co-infected and early co-infected mice [25]. Strongyloides spp infection in the murine model induces a Th2 response and regulatory cytokine induction (IL-10), leading to a suppression of pro-inflammatory cytokines and dimin- ishing Th1 response [26]. These pro-inflammatory cytokines (Th1 response) are present in the acute phase of Chagas disease [27]. Thus, co-infection with different parasites may result in complex interactions, which may lead to altered immunological responses of the host. The relationship between positive T.cruzi RT-PCR in peripheral blood and helminth infec- tion (mostly strongyloidiasis) found in this study provides highly relevant data to better under- stand the role of the PCR in the management of Chagas disease patients. Helminth infection could increase the probability of having a positive T. cruzi PCR. Given that current clinical tri- als that evaluate treatment efficacy in Chagas disease are based on the positivity of T. cruzi PCR, this fact may be relevant. [13, 28]. Further studies are needed to evaluate the impact of treating the helminth infection on the positivity of T. cruzi PCR. Another issue that has to be taken into account is that almost all patients in our study came from Bolivia. The geographical distribution of the different T. cruzi discrete typing units (DTUs) differs from country to country, which may have impact in the clinical presentation or in the proportion of patients with positive T. cruzi PCR in peripheral blood [29]. This study has some limitations. First of all, as we have mentioned previously, the study has been performed with a relatively small number of patients; nevertheless, the study population is representative of Chagas disease patients attended in Spanish tropical medicine units. Sec- ondly, the diagnosis of strongyloidiasis has relied in serological tests in most of the cases. Although serology is not the gold standard for the S.stercoralis infection diagnosis, previous studies have demonstrated its usefulness [30]. New tests based on molecular biology such as PCR could increase the accuracy of helminth infection diagnosis. Finally, T. cruzi PCR was determined only at one point, which may underestimate the kinetics of the parasite. In summary, we observed a high prevalence of S. stercoralis infection among chronic Chagas disease patients attended in our tropical medicine unit. Strongyloidiasis was associated with significantly higher proportion of positive T. cruzi RT-PCR determined in peripheral blood. These data increase the scarce available information to understand the role of PCR techniques in the management of Chagas disease patients. Further studies are needed to deepen and con- firm this interesting relationship. Supporting Information S1 Checklist. STROBE Checklist. (DOCX) Author Contributions Conceived and designed the experiments: FS IM. Performed the experiments: FS ES ASM IM. Analyzed the data: FS ES ASM MMG EC IM. Wrote the paper: FS ES ASM MMG EC IM. References 1. Schmunis GA, Yadon ZE. Chagas disease: a Latin American health problema becoming a world health problem. Acta Trop. 2010; 115: 14–21. doi: 10.1016/j.actatropica.2009.11.003 PMID: 19932071 2. 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