Ecological scenario and Trypanosoma cruzi DTU characterization of a fatal acute Chagas disease case transmitted orally (Espírito Santo state, Brazil)

Dario et al. Parasites & Vectors (2016) 9:477 DOI 10.1186/s13071-016-1754-4 RESEARCH Open Access Ecological scenario and Trypanosoma cruzi DTU characterization of a fatal acute Chagas disease case transmitted orally (Espírito Santo state, Brazil) Maria Augusta Dario1, Marina Silva Rodrigues1, Juliana Helena da Silva Barros1, Samanta Cristina das Chagas Xavier1, Paulo Sérgio D’Andrea2, André Luiz Rodrigues Roque1 and Ana Maria Jansen1* Abstract Background: Trypanosoma cruzi infection via oral route results in outbreaks or cases of acute Chagas disease (ACD) in different Brazilian regions and poses a novel epidemiological scenario. In the Espírito Santo state (southeastern Brazil), a fatal case of a patient with ACD led us to investigate the enzootic scenario to avoid the development of new cases. At the studied locality, Triatoma vitticeps exhibited high T. cruzi infection rates and frequently invaded residences. Methods: Sylvatic and domestic mammals in the Rio da Prata locality, where the ACD case occurred, and in four surrounding areas (Baia Nova, Buenos Aires, Santa Rita and Todos os Santos) were examined and underwent parasitological and serological tests. Triatomines were collected for a fecal material exam, culturing and mini-exon gene molecular characterization, followed by RFLP-PCR of H3/Alul. Paraffin-embedded cardiac tissue of a patient was washed with xylene to remove paraffin and DNA was extracted using the phenol-chloroform method. For genotype characterization, PCR was performed to amplify the 1f8, GPI and 18S rRNA genes. In the case of V7V8 SSU rRNA, the PCR products were molecularly cloned. PCR products were sequenced and compared to sequences in GenBank. Phylogenetic analysis using maximum likelihood method with 1000 bootstrap replicates was performed. Results: None of the animals showed positive hemocultures. Three rodents and two dogs showed signs of infection, as inferred from borderline serological titers. T. vitticeps was the only triatomine species identified and showed T. cruzi infection by DTUs TcI and TcIV. The analysis of cardiac tissue DNA showed mixed infection by T. cruzi (DTUs I, II, III and IV) and Trypanosoma dionisii. Conclusions: Each case or outbreak of ACD should be analyzed as a particular epidemiological occurrence. The results indicated that mixed infections in humans may play a role in pathogenicity and may be more common than is currently recognized. Direct molecular characterization from biological samples is essential because this procedure avoids parasite selection. T. dionisii may under certain and unknown circumstances infect humans. The distribution of T. cruzi DTUS TcIII and TcIV in Brazilian biomes is broader than has been assumed to date. Keywords: Mixed infections, Trypanosoma cruzi DTU, Trypanosoma dionisii, Triatomine, Oral infection, Acute chagas (Continued on next page) * Correspondence: 1 Laboratory of Trypanosomatid Biology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/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://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dario et al. Parasites & Vectors (2016) 9:477 Page 2 of 14 (Continued from previous page) disease Abbreviation: ACD, Acute chagas disease; BLAST, Basic local alignment search tool; CD, Chagas disease; COLTRYP, Coleção de trypanosoma de mamíferos silvestres, domésticos e vetores; DTU, Discrete typing unit; ELISA, Enzyme-Linked Immunosorbent Assay; ES, Espírito Santo state; GPI, Glucose-phosphate isomerase; IFAT, Indirect Immunofluorescent Antibody Test; LIT, Liver Infusion Tryptose; ML, Maximum likelihood; NNN, Novy Mc Neal Nicole; PCR, Polymerase chain reaction; RFLP, Restriction fragment length polymorphism; Sesa/ES, Espírito Santo state Health Department; ZCC, Zoonosis Control Center Background The genus Trypanosoma (Trypanosomatidae, Kinetoplastida), which includes the subgenus Schizotrypanum, is composed of numerous species that are distributed worldwide. Humans or other mammals can serve as suitable hosts. With the exception of Trypanosoma cruzi, other species of this subgenus are restricted to bats. Due to their morphological similarity, these other species have been classically described as T. cruzi-like [1, 2]. The biological cycles of Schizotrypanum trypanosomes are similar, differing only in the identity of their mammalian hosts and their hemipteran vectors. Trypanosoma cruzi marinkellei is transmitted by triatomine insects of the genus Cavernicola, and Trypanosoma dionisii is transmitted by Cimicidae. Species of Schizotrypanum are the only trypanosomes described thus far that infect mammalian cells and multiply inside them as amastigotes [2–4]. There is still much to study regarding T. dionisii and T. c. marinkellei. Furthermore, despite being the subject of intensive study for more than 100 years, there are still several unanswered questions pertaining to the biology of T. cruzi. Trypanosomiasis by T. cruzi is primarily a sylvatic enzooty. This flagellate species is widely distributed in nature, occurring from the southern United States (USA) through southern Argentina and Chile [5]. Trypanosoma cruzi circulates among 150 mammal species and is capable of colonizing almost any tissue of its mammalian hosts. It can also be transmitted by dozens of triatomine species [6]. The parasite transmission cycle is complex in nature because, in addition to its tremendous host species diversity, T. cruzi is highly genetically diverse [7]. Currently, six Discrete Typing Units (DTUs), TcI to TcVI, in addition to TcBat, are recognized [8–10]. Correlations among DTUs/geographical distribution/ host species and pathogenicity are still controversial. Classically, TcII, TcV and TcVI were related to severe human diseases and TcI, TcIII and TcIV were related to the sylvatic cycle [10], but in the Amazon region, Colombia and Venezuela, reports have described human disease by TcI, TcIII and TcIV [11–16]. Although diverse studies have proposed these and other correlations, this topic still requires further clarification. Trypanosoma cruzi populations can be selected when they are grown under laboratory conditions or even when natural infections lead to erroneous conclusions regarding DTU variety and putative associations [17, 18]. Similarly, due to the undersampling of hosts and habitats, the ecology of the DTUs of T. cruzi is far from well understood. (...truncated)