Molecular identification and genotyping of Trypanosoma cruzi DNA in autochthonous Chagas disease patients from Texas, USA

Accepted Manuscript Molecular identification and genotyping of Trypanosoma cruzi DNA in autochthonous Chagas disease patients from Texas, USA

Melissa N Garcia, Hadley Burroughs, Rodion Gorchakov, Sarah M Gunter, Eric Dumonteil, Kristy O Murray, Claudia P Herrera PII: DOI: Reference:

S1567-1348(17)30016-3 doi: 10.1016/j.meegid.2017.01.016 MEEGID 3048

To appear in:

Infection, Genetics and Evolution

Received date: Revised date: Accepted date:

24 August 2016 12 January 2017 14 January 2017

Please cite this article as: Melissa N Garcia, Hadley Burroughs, Rodion Gorchakov, Sarah M Gunter, Eric Dumonteil, Kristy O Murray, Claudia P Herrera , Molecular identification and genotyping of Trypanosoma cruzi DNA in autochthonous Chagas disease patients from Texas, USA. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Meegid(2017), doi: 10.1016/j.meegid.2017.01.016

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Molecular identification and genotyping of Trypanosoma cruzi DNA in autochthonous Chagas disease patients from Texas, USA

T

Melissa N Garciaa, Hadley Burroughsb, Rodion Gorchakova, Sarah M Guntera, Eric Dumonteilb,c,

CR

Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine

US

a

IP

Kristy O Murraya, Claudia P Herrerab*

Department of Tropical Medicine, Vector-Borne Infectious Disease Research Center, Tulane

M

b

AN

and Texas Children’s Hospital, Houston, Texas, United States of America

ED

University, School of Public Health and Tropical Medicine, New Orleans, Louisiana, United

Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Autonomous University of

CE

c

PT

States of America

AC

Yucatan (UADY), Merida, Yucatan, Mexico

* Corresponding author at: Department of Tropical Medicine, Tulane University, School of Public Health and Tropical Medicine, 1440 Canal Street, New Orleans, LA 70112. USA. Tel: +504 2 278 29 69. E-mail address: [email protected]

1

ACCEPTED MANUSCRIPT Abstract The parasitic protozoan Trypanosoma cruzi, the causative agent of Chagas disease, is widely distributed throughout the Americas, from the southern United States (US) to northern Argentina, and infects at least 6 million people in endemic areas. Much remains unknown about

IP

T

the dynamics of T. cruzi transmission among mammals and triatomine vectors in sylvatic and

CR

peridomestic eco-epidemiological cycles, as well as of the risk of transmission to humans in the US. Identification of T. cruzi DTUs among locally-acquired cases is necessary for enhancing our

US

diagnostic and clinical prognostic capacities, as well as to understand parasite transmission cycles. Blood samples from a cohort of 15 confirmed locally-acquired Chagas disease patients

AN

from Texas were used for genotyping T. cruzi. Conventional PCR using primers specific for the

M

minicircle variable region of the kinetoplastid DNA (kDNA) and the highly repetitive genomic

ED

satellite DNA (satDNA) confirmed the presence of T. cruzi in 12/15 patients. Genotyping was based on the amplification of the intergenic region of the miniexon gene of T. cruzi and

PT

sequencing. Sequences were analysed by BLAST and phylogenetic analysis by Maximum Likelihood method allowed the identification of non-TcI DTUs infection in six patients, which

CE

corresponded to DTUs TcII, TcV or TcVI, but not to TcIII or TcIV. Two of these six patients

AC

were also infected with a TcI DTU, indicating mixed infections in those individuals. Electrocardiographic abnormalities were seen among patients with single non-TcI and mixed infections of non-TcI and TcI DTUs. Our results indicate a greater diversity of T. cruzi DTUs circulating among autochthonous human Chagas disease cases in the southern US, including for the first time DTUs from the TcII-TcV-TcVI group. Furthermore, the DTUs infecting human patients in the US are capable of causing Chagasic cardiac disease, highlighting the importance of parasite detection in the population.

2

ACCEPTED MANUSCRIPT Keywords: Chagas Disease; Trypanosoma cruzi; Texas; United States; Epidemiology; Molecular identification; Genotyping; DTU; Discrete Taxonomic Units; Triatoma

1. Introduction

T

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, is an anthropozoonosis

IP

that represents a major public health problem in the Americas, with a burden of disease 7.5 times

CR

higher than malaria as measured by disability-adjusted life-years-DALYs (Bern C, 2015). Although the US was initially defined as non-endemic for Chagas disease, the presence

US

of T. cruzi has now been amply demonstrated as enzootic in different regions of the southern half

AN

of the country, ranging from Georgia to California (Bern et al., 2015; Montgomery et al., 2014). A report estimated that up to 300,000 US residents are infected with T. cruzi, based on the

M

expected seroprevalence in immigrant populations from Latin America (Bern et al., 2011; Bern

ED

and Montgomery, 2009; Cantey et al., 2012; Montgomery et al., 2014). As a consequence, mandatory blood donor screening was initiated in 2007, and now more than 2,000 US blood

PT

donors have tested positive for the parasite that causes Chagas disease (Banks, 2015).

CE

This active blood bank surveillance has created a convenience sampling of U.S. residents with infection, and in particular we have been able to identify T. cruzi-positive blood donors

AC

from the southeastern region of Texas (Garcia et al., 2015a). From that cohort, we previously found that 16 out of 34 (47%) T. cruzi positive blood donors had evidence of locally-acquired transmission, defined as a lack of significant travel history to Chagas endemic areas of Mexico, Central America, or South America, as well as lack of alternative risk factors, including congenital infection or blood transfusion/organ transplantation, thus suggesting autochthonous vector-borne infection (Garcia et al., 2015b, Gunter et al., 2017). To support a significant risk of

3

ACCEPTED MANUSCRIPT infection, studies have reported a high proportion of human blood meals (>50%) in several triatomine species, including ones that also tested positive for T. cruzi parasites, collected in Texas and the southern US (Gorchakov et al., 2016; Klotz et al., 2014; Waleckx, 2014). An autochthonous case of T. cruzi transmission has also been reported in Southern California

T

(Hernandez et al., 2016). Collectively, these findings show that the risk of local vectorial

IP

transmission may have been strongly underestimated.

CR

Due to its high genetic variability, T. cruzi has been classified into six discrete typing units (DTUs) named TcI-TcVI, and a seventh one named Tcbat (Zingales et al., 2012, Lima et

US

al., 2015). Each DTU has been proposed to be associated with different transmission cycles,

AN

hosts, and geographical distributions throughout the Americas (Zingales et al., 2012). Initial reports on the genetic characterization of T. cruzi strains from the US have only demonstrated the

M

presence of TcI and TcIV DTUs (Barnabe et al., 2001; Roellig et al., 2008; Roellig, 2013). Also,

ED

parasites from five autochthonous human cases (from Texas, California, and Louisiana) were previously genotyped as TcI (Roellig et al., 2008). The majority of human infections in Latin

PT

America are caused by TcI, TcII, TcV, and TcVI DTUs, and these are most frequently found

CE

within domestic transmission cycles. Further identification of T. cruzi DTUs among locallyacquired cases is necessary for enhancing our diagnostic and clinical prognostic capacities, as

AC

well as to understand parasite transmission cycles. In the present study, we took advantage of one of the largest known cohorts of 16 previously reported autochthonous human cases of T. cruzi infection from Texas for the genotyping and molecular identification of T. cruzi parasites. Our goal was to better understand the origin and characteristics of strains associated with human transmission in US residents.

4

ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1 Ethics statement This study was approved by the Institutional Review Boards at Baylor College of Medicine, the Gulf Coast Regional Blood Bank, and South Texas Regional Blood and Tissue Centers (H-

T

32321). All case-patients provided written informed consent for the collection of blood samples.

CR

IP

No minors were included in this study.

2.2 Study Population

US

In 2013, we established a cohort of T. cruzi-positive blood donors from the greater Houston and

AN

San Antonio metropolitan areas with autochthonous infection (Garcia et al., 2015a; Gunter et al., 2017)(Table 1). T. cruzi infection was confirmed using multiple assays, including: (1) blood

M

bank screening test by Ortho T. cruzi ELISA (Ortho-Clinical Diagnostics, Raritan, NJ) or Abbott

ED

Prism Chagas (Abbott Laboratories, Abbott Park, IL) and RIPA (Quest Diagnostics, Madison, NJ), (2) Baylor College of Medicine: Stat-pak (Chembio Diagnostic Systems, Inc, Medford,

PT

NY), DPP (Chembio Diagnostic Systems, Inc, Medford, NY), immunofluorescence assay, and

CE

Hemagen Chagas Kit (Hemagen Diagnostics, Inc, Waltham, MA), and (3) U.S. Centers for Disease Control and Prevention-Parasitic Diseases Branch: TESA immunoblot and enzyme

AC

immunoassay. Autochthonous cases were defined as persons who did not have the following traditional risk factors: history of living in an endemic country in Latin America, travel history greater than 2 weeks in an endemic country in Latin America, congenital risk (a mother born/resided in an endemic country in Latin America), evidence of organ transplantation as the source of infection, or evidence of blood transfusion as the source of infection. Precise origin of locally acquired infection was unknown; however, rural residence, occupational exposure, and

5

ACCEPTED MANUSCRIPT regular outdoor recreational activities were commonly reported among the T. cruzi-positive blood donors (Garcia et al., 2015a). Confirmed autochthonous cases of T. cruzi-positive blood donors resided across the greater southeastern and south central region of Texas (Figure 1). As part of the original cohort follow-up, cardiac evaluations were performed on all T.

T

cruzi-positive blood donors. These assessments included an electrocardiogram performed by a

CR

IP

trained technician and interpreted by a licensed physician (Garcia et al., 2015c).

2.3 PCR testing for T. cruzi parasites

US

Of the 16 confirmed cases of locally-acquired T. cruzi infection, blood samples for

AN

genotyping were available for 15 donors. Samples were mixed with an equal volume of 6 M guanidine HCl-0.2M EDTA solution immediately after sample collection. DNA was extracted

M

using DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA). All DNA samples were tested by

ED

PCR for the presence of T. cruzi. One negative blood sample spiked with T. cruzi parasites and one negative blood sample without T. cruzi DNA were included as internal controls for DNA

PT

extraction (Schijman et al, 2011). A no DNA template negative control was also used in the

CE

PCR.

T. cruzi infection was determined by standard PCR using primers Tc121/Tc122 specific

AC

for the minicircle variable region of the kinetoplast DNA (kDNA) (Wincker et al., 1994) and the highly repetitive genomic satellite DNA (satDNA) with the primers TcZ1/TcZ2 (Moser et al., 1989; Virreira et al., 2003). Each sample was also analyzed for the human β-globin fragment to verify DNA integrity and the lack of PCR inhibitors (Virreira et al., 2003). Those samples demonstrating a band at 330bp for kDNA and/or 188 bp for satDNA were considered T. cruzipositive and selected for further genotyping.

6

ACCEPTED MANUSCRIPT 2.4 Molecular identification of T. cruzi DTUs T. cruzi genotyping was performed using the intergenic region of the miniexon gene of T. cruzi using primers reported by Wincker et al. (1994). This technique distinguishes TcI from other DTUs referred to as non-TcI, which include TcII, TcIII, TcIV, TcV and TcVI. In this

T

reaction, TcI has an expected fragment size of 220 bp and non-TcI DTUs have an expected

IP

fragment size of 130 bp. Alternative primers amplifying a different region of the miniexon gene

CR

were also tested (Fernandes O, 2001; Souto R, 1996). Samples determined to be non-TcI were further analyzed by PCR amplification of the D7 divergent domain of the 24Sα rRNA gene and

US

the size-variable domain of the 18S rRNA gene. The primers used were D71/D72 and V1/V2,

AN

respectively (Souto et al., 1993; Clark & Pung, 1994).

M

2.5 Sequencing and editing of the miniexon gene intergenic region

ED

DTU identification was confirmed and further refined by sequencing PCR amplicons from the miniexon intergenic region after purification using a PureLink Quick PCR purification kit

PT

(Thermo Scientific, Waltham, MA). For samples that presented more than one amplification

CE

band for a molecular marker, each band was excised and recovered using Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. All

AC

samples were sequenced by GENEWIZ, Inc. (South Plainfield, NJ). Sequences were analysed by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for a preliminary parasite DTU confirmation. The sequences were aligned and edited manually using MEGA version 5 software (Tamura et al., 2011).

2.6 Phylogenetic analysis

7

ACCEPTED MANUSCRIPT A total of eight sequences of sufficient quality (GenBank accession numbers: KU883256KU883263) were obtained from 6 autochthonous human samples for phylogenetic analysis. Additionally, 25 reference sequences covering the six DTUs were obtained from GenBank database. The reference sequences were from strains MN (Accession number: AY367128.1),

T

Sc43 (AY367127.1), Tu18 (AY367125.1), CL (U57984.1), CANIII (AY367123.1), M5631

IP

(AY367126), M6241 (AF050522), USAOPOSSUMcl2 (JQ581510.1), 92090802Pcl1 USA

CR

(JQ581481.1), JEMC (EU127299) and sequences from New Orleans T. cruzi DNA (KM376435KM376449). The phylogenetic tree was inferred using the Maximum Likelihood (ML) method.

US

The best fit substitution model selected for this data set was determined to be GTR (general time-

AN

reversible model) + I (invariable sites proportion) + G (a gamma disturbed rate of variation among sites). The tree and the robustness of the nodes were evaluated by bootstrap on 1000

M

replications by heuristic search. The best tree was obtained automatically by applying Neighbor-

ED

Join and BioNJ algorithms and then selecting the topology with superior log likelihood value

PT

using MEGA version 5 software (Tamura et al., 2011).

CE

3. Results

3.1 PCR Identification and genotyping of T. cruzi

AC

Twelve of 15 (80%) donors” blood samples were confirmed as T. cruzi positive by conventional PCR using the kinetoplastid DNA (kDNA) and the highly repetitive genomic satellite DNA (satDNA). The kDNA marker on PCR positive samples was found to be more sensitive (12/12; 100%) than the satDNA marker (9/12; 75%). The genotype of T. cruzi was determined using the miniexon gene marker using primers from Virreira et al. (2006). From the 12 PCR-positive samples, differentiation among DTUs was

8

ACCEPTED MANUSCRIPT achieved in a total of 6 donors (Supplementary figure 1). Infection with non-TcI DTUs (TcIITcIII-TcIV-TcV-TcVI) were detected in all 6 donors. In addition, co-infections with TcI were detected in 2/6 (33.3%) donors. Alternative primers (Fernandes O, 2001; Souto R, 1996) gave negative results for most or all samples tested. These samples were further analyzed using

T

ribosomal markers to specify the infecting non-TcI DTU, and unfortunately, negative results

CR

IP

were obtained for all the samples.

3.2 Mini-exon intergenic region sequencing and phylogenetic analysis

US

DTU identification was confirmed and refined by sequencing of the mini-exon intergenic

AN

region and phylogenetic analysis on a total of 8 sequences from the autochthonous cases. The analysis involved 31 nucleotide sequences with a length of 185 bp. All sequence positions

M

containing gaps and missing data were eliminated. To avoid artefactual gaps, non-TcI and TcI

ED

sequences were analyzed separately. The un-rooted phylogenetic trees of non-TcI samples (Figure 2) allowed the partial resolution of the non-TcI DTUs with significant bootstrap values

PT

of the ML analysis (more than 60%). Indeed, four main clusters which included T. cruzi

CE

sequences from the six donors (006-TX, 007-TX, 044-TX, 046-TX, 047-TX, 050-TX) and reference strains (including some from the US) were identified.

AC

The first cluster including six sequences from donor samples was part of a larger cluster corresponding to the DTUs TcII-TcV-TcVI, with a significant bootstrap of 99%, but these three DTUs could not be resolved any further with the available sequence data. A second cluster of reference sequences corresponded to TcIV DTU with a bootstrap of 100%, and a third cluster included reference sequences for the TcIII DTU. However, none of our human samples clustered with TcIII or TcIV.

9

ACCEPTED MANUSCRIPT Analysis of sequences corresponding to TcI confirmed that the two sequences were closely related to other TcI reference sequences (data not shown).

3.3 Cardiac Assessments and Correlates to Identified DTU

T

Electrocardiogram (ECG) abnormalities of donors with single and mixed DTU infections

IP

were mostly minor (Table 2). However, 2/2 of the donors with mixed TcI- non-TcI infections

CR

presented with abnormalities, while only 2/4 of the donors with single non-TcI infection did so, suggesting that mixed infections may lead to more frequent ECG alterations, but due to the small

AN

US

sample size, no statistical inferences could be made.

4. Discussion

M

The understanding of T. cruzi transmission and Chagas disease clinical outcomes is

ED

complicated by the presence of six discrete typing units (DTUs) that vary greatly according to the geographic region (Herrera et al., 2007; Herrera et al., 2009; Llewellyn et al., 2009; Spotorno

PT

A, 2008; Zingales et al., 2009). Our study is the first to describe the DTUs circulating among one

CE

of the largest known cohorts of locally-acquired human cases in the southern US. T. cruzi infection was confirmed by PCR in 12/15 patients, in agreement with the expected sensitivity of

AC

this method (Schijman et al., 2011; Virreira et al., 2003). Both single and mixed infections of TcI and non-TcI DTUs were present in these cases. These data showed for the first time the presence of TcII-TcV-TcVI DTUs in human samples in the US, as well as the presence of mixed infections of TcI with TcII-TcV-TcVI DTUs in autochthonous cases of Chagas disease in Texas. Genetic diversity of circulating T. cruzi DTUs among North American mammalian reservoirs have been reported in previous studies. Both TcI and TcIV had been reported in

10

ACCEPTED MANUSCRIPT triatomine vectors, wildlife species, and TcI in five human cases from the southern US (Charles et al., 2013; Roellig et al., 2008; Shender et al., 2016). Identification of TcI DTU in our human cohort was thus expected considering that it is the dominant DTU in Mexico and has been previously detected in the US. TcI has been associated with chronic Chagasic cardiac disease in

T

several regions (Bosseno et al., 2002; Roellig et al., 2013). However, the presence of non-TcI

IP

strains corresponding to TcII, TcV or TcVI in our human cohort as well as the absence of TcIV

CR

were unexpected observations. Nonetheless, it is in agreement with the recent identification of TcII DTU in small rodents captured close to the residence of an autochthonous human case in a

US

rural area in New Orleans (Herrera et al., 2015; López-Cancino et al., 2015). Also, while we

AN

were unable to resolve TcII, TcV and TcVI DTUs with our molecular marker, these observations suggest that TcII may be the most likely DTU circulating in the autochthonous cases described

M

here. This is further supported by the apparent lack of detection of TcIII, TcV, and TcVI DTUs

ED

in the US in previous studies, and that we did not identify TcIII and TcIV in our molecular analysis. Indeed, TcII, TcV, and TcVI DTUs are genetically closely related, requiring more

PT

complex methodologies for distinction of individual strains (Monje-Rumi et al., 2015).

CE

Unfortunately, the alternative primers for the miniexon gene and ribosomal markers that we tested were unable to provide reliable amplifications, likely due to a low amount of T. cruzi DNA

AC

present in the blood of the donors, suggesting that the method used was more sensitive. Mixed infections with more than one DTU have been commonly reported from South American countries (Abolis et al., 2011; Monje-Rumi et al., 2015), but this study is the first report of human co-infections originating in the US. Triatomines in the US were previously found to be infected with both TcI and TcIV DTUs, suggesting that a potential for mixed infections in humans might exist (Barnabé et al., 2001; Roellig et al., 2008).

11

ACCEPTED MANUSCRIPT Minor ECG abnormalities often found with Chagasic cardiac disease were common and present in patients with both single and mixed T. cruzi infections, although they tended to be more frequent in those with mixed infections. Parasite DTUs have been linked with differences in tissue tropism and in the development of clinical manifestations, although some studies have

T

failed to detect clear associations (Gonzalez et al., 2010; Martinez-Perez et al., 2016). In

IP

Argentina, infection with TcII-TcV-TcVI DTUs was found to be more frequent in chronic

CR

Chagas heart disease patients compared to asymptomatic patients (Cura et al., 2012), suggesting that these DTUs may lead to more severe disease. A high risk of developing Chagasic

US

cardiomyopathy may thus exist for autochthonous cases in the US. Regardless, future studies that

AN

include a larger sample size is required for the detection of associations among DTUs and

M

cardiac manifestations.

ED

5. Conclusion

In conclusion, our findings highlight the presence of multiple T. cruzi DTUs circulating

PT

among autochthonous human Chagas disease cases in Texas, including for the first time

CE

detection of DTUs from the TcII-TcV-TcVI group. Our study indicates that residents with locally-acquired T. cruzi infection are at risk for acquiring infection with DTUs associated with

AC

the development of Chagasic cardiac disease. Furthermore, our findings support the need to identify, diagnose, and treat autochthonous Chagas disease in the US. Finally, a better understanding of circulating DTUs in vectors and mammalian reservoirs in the country is crucial to identify potential sources of infection, which is necessary to develop tailored public health interventions. Therefore, our results have important epidemiological and clinical implications that warrant future investigations.

12

ACCEPTED MANUSCRIPT Acknowledgements The authors would like to thank Dr. Pierre Buekens (Dean, School of Public Health and Tropical Medicine - Tulane University) for supporting this study. This project was funded in part by a private donor at Texas Children’s Hospital, Houston, Texas and by a pilot grant endowed by

T

Baylor College of Medicine Cardiovascular Research Institute and internal funding from the

IP

School of Public Health and Tropical Medicine- Tulane University. Also we thank Dr. Dawn

CR

Wesson (SPHTM - Tulane University) for her critical revisions of the manuscript, and Henry

US

Provonost and Alice Belloc (SPHTM - Tulane University) for their technical support.

AN

References

Abolis, N.G., Araújo, S.M., Toledo, M.J., Fernandez, M.A., Gomes, M.L., 2011. Trypanosoma

M

cruzi I-III in southern Brazil causing individual and mixed infections in humans, sylvatic

ED

reservoirs and triatomines. Acta Trop 120, 167-172.

Afonso, A.M., Ebell, M.H., Tarleton, R.L., 2012. A systematic review of high quality diagnostic

PT

tests for Chagas disease. PLoS Negl Trop Dis 6, e1881.

CE

American Association of Blood Banks’s Biovigilance Network. 2008. Available at:

AC

http://www.aabb.org/research/hemovigilance/Pages/chagas.aspx. Accessed 20 December 2015.

Barnabe, C., Yaeger, R., Pung, O., Tibayrenc, M., 2001. Trypanosoma cruzi: A considerable phylogenetic divergence indicates that the agent of Chagas disease is indigenous to the native fauna of the United States. Exp Parasitol 99: 73-79. Bern C, 2015. Chagas’ Disease. N Engl J Med. 373: 456-66.

Bern C, Montgomery SP (2009) An estimate of the burden of Chagas disease in the United States. Clin Infect Dis 49: e52-54.

13

ACCEPTED MANUSCRIPT Bosseno, M.F., Barnabé, C., Magallón Gastélum, E., Lozano Kasten, F., Ramsey, J., Espinoza, B., Brenière, S.F., 2002. Predominance of Trypanosoma cruzi lineage I in Mexico. J Clin Microbiol 40, 627-632.

Burgos, J.M.D., M. Vigliano, C. Bisio, M. Risso, M. Duffy, T. Cura, C. Brusses, B. Favaloro, L. Leguizamon, M. S. Lucero, R. H. Laguens, R. Levin, M. J. Favaloro, R. Schijman, A. G., 2010.

T

Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas

CR

IP

heart disease and reactivation after heart transplantation. Clin Infect Dis 51, 485-495.

Cantey, P.T., Stramer, S.L., Townsend, R.L., Kamel, H., Ofafa, K., Todd, C.W., Currier, M.,

US

Hand, S., Varnado, W., Dotson, E., Hall, C., Jett, P.L., Montgomery, S.P., 2012. The United States Trypanosoma cruzi Infection Study: evidence for vector-borne transmission of the parasite

AN

that causes Chagas disease among United States blood donors. Transfusion 52, 1922-1930. Chagas disease in Latin America: an epidemiological update based on 2010 estimates. Wkly

M

Epidemiol Rec 2015 90: 33-43.

ED

Charles, R.A., Kjos, S., Ellis, A.E., Barnes, J.C., Yabsley, M.J., 2013. Southern plains woodrats (Neotoma micropus) from southern Texas are important reservoirs of two genotypes of

PT

Trypanosoma cruzi and host of a putative novel Trypanosoma species. Vector Borne Zoonotic

CE

Dis 13, 22-30.

Clark CG, Pung OJ. Host specificity of ribosomal DNA variation in sylvatic Trypanosoma cruzi

AC

from North America. Mol Biochem Parasitol. 1994;66(1):175–9.

Cura, C.I., Lucero, R.H., Bisio, M., Oshiro, E., Formichelli, L.B., Burgos, J.M., Lejona, S., Bruses, B.L., Hernandez, D.O., Severini, G.V., Velazquez, E., Duffy, T., Anchart, E., Lattes, R., Altcheh, J., Freilij, H., Diez, M., Nagel, C., Vigliano, C., Favaloro, L., Favaloro, R.R., Merino, D.E., Sosa-Estani, S., Schijman, A.G., 2012. Trypanosoma cruzi discrete typing units in Chagas disease patients from endemic and non-endemic regions of Argentina. Parasitology 139, 516521.

14

ACCEPTED MANUSCRIPT Fernandes, O., Santos, S.S., Cupolillo, E., Mendonca, B., Derre, R., Junqueira, A.C., Santos, L.C., Sturm, N.R., Naiff, R.D., Barret, T.V., Campbell, D.A., Coura, J.R., 2001. A mini-exon multiplex polymerase chain reaction to distinguish the major groups of Trypanosoma cruzi and T. rangeli in the Brazilian Amazon. Trans R Soc Trop Med Hyg 95, 97-99.

Garcia, M., Aguilar, D., Gorchakov, R., Rossmann, S., Montgomery, S., Rivera, H., Woc-

T

Colburn, L., Hotez, P., Murray, K., 2015a. Evidence of autochthonous Chagas disease in

CR

IP

southeastern Texas. Am J Trop Med Hyg 92, 325-330.

Garcia, M., Murphy, S., Gross, A., Wagner, J., Murray, K., 2015b. Knowledge, attitudes, and

AN

causes Chagas disease. Parasit Vectors 8, 197.

US

practices of Texas hunters: a potentially high-risk population for exposure to the parasite that

Garcia, M., Murray, K., Hotez, P., Rossmann, S., Gorchakov, R., Ontiveros, A., Woc-Colburn,

M

L., Bottazzi, M., Rhodes, C., Ballantyne, C., Aguilar, D., 2015c. Development of Chagas cardiac

ED

manifestations among Texas blood donors. Am J Cardiol 115, 113-117.

Gonzalez CI, Ortiz S, Solari A (2010) Colombian Trypanosoma cruzi major genotypes

PT

circulating in patients: minicircle homologies by cross-hybridization analysis. Int J Parasitol 40:

CE

1685-1692.

Gorchakov, R., Trosclair, L.P., Wozniak, E.J., Feria, P.T., Garcia, M.N., Gunter, S.M., Murray,

AC

K.O., 2016. Trypanosoma cruzi infection prevalence and bloodmeal analysis in triatomine vectors of Chagas disease from rural peridomestic Locations in Texas, 2013-2014. J Med Entomol 911-918.

Gunter, S.M., Murray, K.O., Gorchakov, R., Beddard, R., Montgomery, S.P., Rivera, H., Brown, E.L., Widman, L., Aguilar, D., Garcia, M.N., 2017. Likely autochthonous transmission of Trypanosoma cruzi to humans, Southcentral Texas, USA. Emerg Infect Dis In press.

15

ACCEPTED MANUSCRIPT Herrera, C., Bargues, M.D., Fajardo, A., Montilla, M., Triana, O., Vallejo, G.A., Guhl, F., 2007. Identifying four Trypanosoma cruzi I isolate haplotypes from different geographic regions in Colombia. Infect Genet Evol 7, 535-539.

Herrera, C., Guhl, F., Falla, A., Fajardo, A., Montilla, M., Adolfo Vallejo, G., Bargues, M.D.,

IP

Colombia based on miniexon gene sequences. J Parasitol Res 2009, 1.

T

2009. Genetic variability and phylogenetic relationships within Trypanosoma cruzi I isolated in

CR

Herrera, C.P., Licon, M.H., Nation, C.S., Jameson, S.B., Wesson, D.M., 2015. Genotype diversity of Trypanosoma cruzi in small rodents and Triatoma sanguisuga from a rural area in

US

New Orleans, Louisiana. Parasit Vectors 8, 123.

Klotz, S.A., Schmidt, J.O., Dorn, P.L., Ivanyi, C., Sullivan, K.R., Stevens, L., 2014. Free-

American journal of medicine 127, 421-426.

AN

roaming kissing bugs, vectors of Chagas disease, feed often on humans in the Southwest. The

M

Lima L, Espinosa-Álvarez O, Ortiz PA, Trejo-Varón JA, Carranza JC, Pinto CM, Serrano MG, Buck GA, Camargo EP, Teixeira MM. 2015 Genetic diversity of Trypanosoma cruzi in bats, and

ED

multilocus phylogenetic and phylogeographical analyses supporting Tcbat as an independent DTU

PT

(discrete typing unit). Acta Trop. 151, 166-177.

Llewellyn, M.S., Miles, M.A., Carrasco, H.J., Lewis, M.D., Yeo, M., Vargas, J., Torrico, F.,

CE

Diosque, P., Valente, V., Valente, S.A., Gaunt, M.W., 2009. Genome-Scale Multilocus Microsatellite Typing of Trypanosoma cruzi Discrete Typing Unit I Reveals Phylogeographic

AC

Structure and Specific Genotypes Linked to Human Infection. PLoS Pathog 5(5): e1000410.

López-Cancino S, Tun-ku.E., De la Cruz-Felix H, Ibarra-Cerdeña C, Izeta-Alberdi A, PechMay A, Mazariegos-Hidalgo C, Valdez-Tah A, Ramsey J, 2015. Landscape ecology of Trypanosoma cruzi in the southern YucatanPeninsula. Acta trop. 151, 58-72.

Martinez-Perez, A., Poveda, C., Ramirez, J.D., Norman, F., Girones, N., Guhl, F., MongeMaillo, B., Fresno, M., Lopez-Velez, R., 2016. Prevalence of Trypanosoma cruzi's Discrete Typing Units in a cohort of Latin American migrants in Spain. Acta trop. 157, 145-150.

16

ACCEPTED MANUSCRIPT Monje-Rumi, M.M., Brandán, C.P., Ragone, P.G., Tomasini, N., Lauthier, J.J., Alberti D'Amato, A.M., Cimino, R.O., Orellana, V., Basombrío, M.A., Diosque, P., 2015. Trypanosoma cruzi diversity in the Gran Chaco: mixed infections and differential host distribution of TcV and TcVI. Infect Genet Evol 29, 53-59.

Montgomery, S.P., Starr, M.C., Cantey, P.T., Edwards, M.S., Meymandi, S.K., 2014. Neglected

T

parasitic infections in the United States: Chagas disease. Am J Trop Med Hyg 90, 814-818.

IP

Moser, D.R., Kirchhoff, L.V., Donelson, J.E., 1989. Detection of Trypanosoma cruzi by DNA

CR

amplification using the polymerase chain reaction. J Clin Microbiol 27, 1477-1482. Porras, A.I., Yadon, Z.E., Altcheh, J., Britto, C., Chaves, G.C., Flevaud, L., Martins-Filho, O.A.,

US

Ribeiro, I., Schijman, A.G., Shikanai-Yasuda, M.A., Sosa-Estani, S., Stobbaerts, E., Zicker, F., 2015. Target Product Profile (TPP) for Chagas Disease Point-of-Care Diagnosis and Assessment

AN

of Response to Treatment. PLoS Negl Trop Dis 9, e0003697.

M

Ramirez, J.D., Guhl, F., Rendon, L.M., Rosas, F., Marin-Neto, J.A., Morillo, C.A., 2010. Chagas Cardiomyopathy Manifestations and Trypanosoma cruzi Genotypes Circulating in Chronic

ED

Chagasic Patients. PLoS Negl Trop Dis 4, e899.

PT

Roellig, D.M., Brown, E.L., Barnabe, C., Tibayrenc, M., Steurer, F.J., Yabsley, M.J., 2008.

CE

Molecular typing of Trypanosoma cruzi isolates, United States. Emerg Infect Dis 14, 1123-1125.

Roellig, D.M., Savage, M.Y., Fujita, A.W., Barnabe, C., Tibayrenc, M., Steurer, F.J., Yabsley,

AC

M.J., 2013. Genetic variation and exchange in Trypanosoma cruzi isolates from the United States. PloS One 8, e56198.

Schijman, A.G., Bisio, M., Orellana, L., Sued, M., Duffy, T., Mejia Jaramillo, A.M., Cura, C., Auter, F., Veron, V., Qvarnstrom, Y., Deborggraeve, S., Hijar, G., Zulantay, I., Lucero, R.H., Velazquez, E., Tellez, T., Sanchez Leon, Z., Galvao, L., Nolder, D., Monje Rumi, M., Levi, J.E., Ramirez, J.D., Zorrilla, P., Flores, M., Jercic, M.I., Crisante, G., Anez, N., De Castro, A.M., Gonzalez, C.I., Acosta Viana, K., Yachelini, P., Torrico, F., Robello, C., Diosque, P., Triana Chavez, O., Aznar, C., Russomando, G., Buscher, P., Assal, A., Guhl, F., Sosa Estani, S.,

17

ACCEPTED MANUSCRIPT DaSilva, A., Britto, C., Luquetti, A., Ladzins, J., 2011. International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. PLoS Negl Trop Dis 5, e931.

Shender, L.A., Lewis, M.D., Rejmanek, D., Mazet, J.A., 2016. Molecular Diversity of Trypanosoma cruzi detected in the vector Triatoma protracta from California, USA. PLoS Negl

T

Trop Dis 10, e0004291.

IP

Souto RP, Zingales B. Sensitive detection and strain classification of Trypanosoma cruzi by

CR

amplification of a ribosomal RNA sequence. Mol Biochem Parasitol. 1993;62:45–52. Souto R, F.O., Macedo A, Campbell D, Zingales B., 1996. DNA markers define two major

US

phylogenetic lineages of Trypanosoma cruzi. Mol Biochem Parasitol 83, 141-152.

AN

Spotorno A, C.L., Solari A., 2008. Differentiation of Trypanosoma cruzi I subgroups through

M

characterization of cytochrome b gene sequences. Infect Genet Evol. 8, 898-900.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5:

ED

molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and

PT

maximum parsimony methods. Mol Biol Evol 28, 2731-2739.

Virreira, M., Torrico, F., Truyens, C., Alonso-Vega, C., Solano, M., Carlier, Y., Svoboda, M.,

CE

2003. Comparison of polymerase chain reaction methods for reliable and easy detection of

AC

congenital Trypanosoma cruzi infection. Am. J. Trop. Med. Hyg 68, 574-582.

Virreira, M, Alonso-Vega, C, Solano M, Jijena J, Brutus L, Bustamante Z, Truyens C, Schneider D, Torrico F, Carlier Y, Svoboda M. 2006. Congenital Chagas disease in bolivia is not associated with DNA polymorphism of Trypanosoma cruzi. Am. J. Trop. Med. Hyg., 75, 871– 879

Waleckx, E.S., J. Richards, B. Dorn, P. L., 2014. Triatoma sanguisuga blood meals and potential for Chagas disease, Louisiana, USA. Emerg Infect Dis 20, 2141-2143.

18

ACCEPTED MANUSCRIPT Wincker P, Britto C, Pereira JB, Cardoso MA, Oelemann W, Morel CM, 1994. Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area. Am J Trop Med Hyg 51: 771-777.

Zingales, B., Andrade, S.G., Brinones, M.R.S., Campbell, D.A., Chiari, E., Fenandes, O., Guhl,

T

F., Lages-Silva, E., Macedo, A.M., Machado, C.R., Miles, M.A., Romanha, A.J., Sturm, N.R.,

IP

Tibayrenc, M., Schijman, A.G., 2009. A new consensus for Trypanosoma cruzi intraspecific

CR

nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz 104

US

(7), 1051-1054.

Zingales, B., Miles, M.A., Campbell, D.A., Tibayrenc, M., Macedo, A.M., Teixeira, M.M.G.,

AN

Schijman, A.G., Llewellyn, M.S., Lages-Silva, E., Machado, C.R., Andrade, S.G., Sturm, N.R., 2012. The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological

AC

CE

PT

ED

M

relevance and research applications. Infect Genet Evol 12, 240-253.

19

ACCEPTED MANUSCRIPT Table 1: Origin of the samples Reference

Reference ID*

(Garcia et al., 2015a)

Case 3 in Case Series

006-TX

(Garcia et al., 2015a)

Case 1 in Case Series

007-TX

(Garcia et al., 2015a)

Case 4 in Case Series

012-TX

(Garcia et al., 2015b)

No assigned case number

026-TX

(Gunter et al.,2017)

Case 5 in Case Series

041-TX

(Gunter et al.,2017)

042-TX

(Gunter et al.,2017)

044-TX

(Gunter et al.,2017)

046-TX

(Gunter et al.,2017)

047-TX

(Gunter et al.,2017)

048-TX

(Gunter et al.,2017)

050-TX

(Gunter et al.,2017)

Case 6 in Case Series

053-TX

(Gunter et al.,2017)

Case 7 in Case Series

056-TX

(Gunter et al.,2017)

Case 9 in Case Series

059-TX

(Gunter et al.,2017)

Case 10 in Case Series

CR

IP

T

004-TX

PT

ID

Case 1 in Case Series

Case 2 in Case Series Case 3 in Case Series Case 4 in Case Series Case 5 in Case Series

AC

CE

ED

M

AN

US

Case 12 in Case Series

* ID as originally published

20

ACCEPTED MANUSCRIPT Table 2. Electrocardiogram findings stratified by T. cruzi DTU Patient ID

T. cruzi DTU

ECG findings Sinus rhythm with 1st degree

006-TX

TcI with TcII-TcV-TcVI

atrioventricular block; left axis

TcI with TcII-TcV-TcVI

Left axis deviation

IP

046-TX

T

deviation

TcII-TcV-TcVI

atrioventricular block; Inferior-

US

007-TX

CR

Sinus rhythm with 1st degree

posterior infarct; T-wave abnormality

TcII-TcV-TcVI

047-TX

TcII-TcV-TcVI

Normal

050-TX

TcII-TcV-TcVI

1st degree atrioventricular block

Normal

AC

CE

PT

ED

M

AN

044-TX

21

ACCEPTED MANUSCRIPT FIGURE LEGENDS

Fig 1. Residential location of Chagas positive blood donors with locally acquired infection. Map of Southeast Texas showing Houston and San Antonio cities. In red the sites where

CR

Fig 2. Phylogenetic analysis of non-TcI sequences.

IP

T

autochthonous human cases were identified using serological screening.

The phylogram depicts the phylogenetic relationships among six non-TcI Trypanosoma cruzi

US

DNA sequences recovered from the blood of six locally acquired T. cruzi infection from Texas,

AN

compared with reference T. cruzi sequences from GenBank (See methods), based on Miniexon intergenic gene sequencing. Bootstrap values appear on each clustering branch. In blue single

ED

M

non-TcI (TcII-V-VI) infections. In green mixed infections TcI- non-TcI (TcII-TcV-TcVI).

PT

Supplementary Fig 1. T. cruzi genotyping by multiplex PCR

A multiplex PCR targeting miniexon gen, using the primers described in Virreira et al. (2006), allows to

CE

discriminate T. cruzi genotypes (or DTU for discrete type unit) present in blood samples as TcI and non-

AC

TcI (comprising TcII to TcVI DTUs) by giving amplicons of different size. Primers MV1 (5’-CAG CGC CAC AGA AAG TGT T-3’) and MV2 (5’-GCT CCT TCA TGT TTG TGT CG-3’) amplify 220 bp of the mini-exon gene from TcI parasites, while MV3 (5’-CTT CCT GTG TTT TCC GGT GT-3’) and MV4 (5’ACA CTG GGG AGG GGT CAG-3’) primers amplify 130 bp from non-TcI parasites. Lanes 1-11: samples from patients. Lane (+): Positve control (Mixture of DNA from Sylvio-X10 strain (TcI) and Esmeraldo strain (non-TcI)). Lanes (-): Negative controls corresponding to a negative blood sample and to a no DNA template PCR control, respectively. Note a band of 130 bp corresponding to non-TcI DTU in lanes 4, 6, 7, 8, 10 and 11, as well as a mixed infection with Tc1 in lane 11.

22

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

Fig. 1

23

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

M

Fig. 2

24

ACCEPTED MANUSCRIPT

Highlights

Trypanosoma cruzi discrete typing units TcI and TcII, TcV or TcVI, but not TcIII or

Co-infection with more than one Trypanosoma cruzi discrete typing unit was identified in Texas-acquired human Chagas disease patients.

CE

PT

ED

M

AN

US

Texas Trypanosoma cruzi discrete typing units can cause cardiac disease.

AC



CR



IP

TcIV were identified in Texas autochthonous human patients.

T



25