Chagas

REVIEW
crossm
Chagas Disease in the United States: a Public Health

Approach
Caryn Bern,a Louisa A. Messenger,b Jeffrey D. Whitman,a James H. Maguirec
a University of California San Francisco School of Medicine, San Francisco, California, USA
b London School of Hygiene and Tropical Medicine, London, UK
c
Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
BIOLOGY AND TRANSMISSION OF TRYPANOSOMA CRUZI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Routes of Transmission. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Vector-borne transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Congenital transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Blood-borne transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Organ-derived transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Oral transmission. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
EPIDEMIOLOGY AND ECOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Global Burden of Chagas Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Triatomine Vector Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Triatomine Distribution in the United States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Allergic Reactions to Triatomine Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Wild and Domestic Animal Reservoirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Wildlife reservoirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Canine Chagas disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Transmission Potential in the United States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
MOLECULAR EPIDEMIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Trypanosoma cruzi Genotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Trypanosoma cruzi Molecular Epidemiology in the United States . . . . . . . . . . . . . . . . . . . . . . . . 11
Issues Underlying T. cruzi Genotyping Data Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
CLINICAL MANIFESTATIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Acute T. cruzi Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Chronic T. cruzi Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Chagas Disease in the Immunocompromised Host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Organ-derived infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Reactivation in cardiac transplant recipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Reactivation of Chagas disease in HIV-coinfected patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
DIAGNOSTIC TECHNIQUES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Molecular Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Diagnostic Serology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
HCT/P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Histopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
ETIOLOGICAL TREATMENT AND CLINICAL MANAGEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Antitrypanosomal Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Acute and Congenital T. cruzi Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Treatment of Chronic T. cruzi Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Management of the Immunocompromised Host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
HUMAN CHAGAS DISEASE IN THE UNITED STATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Citation Bern C, Messenger LA, Whitman JD,
Disease Burden among Latin American Immigrants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Maguire JH. 2020. Chagas disease in the United
Autochthonous Vector-Borne Transmission to Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
States: a public health approach. Clin Microbiol
Blood Donor Screening and Transfusion Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Rev 33:e00023-19. https://doi.org/10.1128/CMR
Organ Donor-Derived Transmission and Organ Donor Screening . . . . . . . . . . . . . . . . . . . . . . . . 25
.00023-19.
Chagas Cardiomyopathy and Heart Transplantation for Chagas Heart Disease in the
United States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Copyright © 2019 American Society for
Congenital Chagas Disease in the United States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Microbiology. All Rights Reserved.
(continued) Address correspondence to Caryn Bern,
Caryn.Bern2@ucsf.edu.
Published 27 November 2019
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Bern et al. Clinical Microbiology Reviews
SPECIAL CONSIDERATIONS IN THE UNITED STATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Physician Awareness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Patient Awareness and Access . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Diagnostic Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
FDA Approval of Benznidazole and Resulting Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Public Health Surveillance and Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
PUBLIC HEALTH APPROACHES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
AUTHOR BIOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
SUMMARY Trypanosoma cruzi is the etiological agent of Chagas disease, usually

transmitted by triatomine vectors. An estimated 20 to 30% of infected individuals
develop potentially lethal cardiac or gastrointestinal disease. Sylvatic transmission cy-
cles exist in the southern United States, involving 11 triatomine vector species and
infected mammals such as rodents, opossums, and dogs. Nevertheless, imported
chronic T. cruzi infections in migrants from Latin America vastly outnumber locally
acquired human cases. Benznidazole is now FDA approved, and clinical and public
health efforts are under way by researchers and health departments in a number of
states. Making progress will require efforts to improve awareness among providers
and patients, data on diagnostic test performance and expanded availability of con-
firmatory testing, and evidence-based strategies to improve access to appropriate
management of Chagas disease in the United States.
KEYWORDS Chagas disease, triatomine, Trypanosoma cruzi, United States
INTRODUCTION
T rypanosoma cruzi is the causative agent of Chagas disease (1, 2). Infection is lifelong
without treatment; thus, prevalence can be high despite low incidence. Current
estimates of 6 million infections and 1.2 million cases of cardiomyopathy place Chagas
disease first in disease burden among parasitic diseases in the Americas (3, 4). Trypano-
soma cruzi organisms are transmitted when infected vector feces enter the bite site or
mucous membranes of a mammalian host. Transmission can also occur through blood
component transfusion, organ transplantation, consumption of food or beverages
contaminated by the vector or vector feces, and in utero from mother to fetus (5).
The classic setting for Chagas disease is rural Latin America, where adobe houses
and the presence of domestic animals favor domestic and peridomestic vector infes-
tation (2). However, transmission in many rural areas has decreased due to vector
control programs, and infected individuals have migrated to Latin American cities (6),
the United States, and Europe (7, 8). Unlike Europe, the United States has well-described
enzootic T. cruzi transmission, involving 11 triatomine species and a range of mamma-
lian hosts (9). Nevertheless, the vast majority of T. cruzi-infected individuals in the
United States are Latin American immigrants infected in their countries of origin. We
will review clinical, epidemiological, and public health aspects of Chagas disease in the
United States, with a focus on the most recent relevant publications.
BIOLOGY AND TRANSMISSION OF TRYPANOSOMA CRUZI

In 1909, Carlos Chagas, a young physician working in rural Brazil, demonstrated the
etiological agent, its vector, several of its reservoir hosts, and the salient manifestations
of the disease that now bears his name, a feat unrivaled in medical history (10). He
named the parasite in honor of his mentor, Oswaldo Cruz (11). Chagas proceeded to
isolate T. cruzi from the blood of a domestic cat and, finally, from a symptomatic
toddler. This “first patient” remained infected for life but never developed chronic
manifestations of Chagas disease and died at 73 from unrelated causes (12). Finally,
Chagas fulfilled Koch’s postulates by reproducing the infection experimentally in
laboratory animals (11).
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Chagas Disease in the United States Clinical Microbiology Reviews
FIG 1 Trypanosoma cruzi morphological forms. (A) The replicating epimastigote form in culture (Giemsa stain). (B) Trypomastigote in a
peripheral blood smear from a patient with acute Chagas disease (Giemsa stain). (C) Nest of amastigotes within a cardiac myocyte in a
patient with chronic Chagas disease (hematoxylin and eosin stain). Courtesy of the Division of Parasitic Diseases and Malaria, U.S. Centers
for Disease Control and Prevention.
In the years since 1909, the life cycle has been more fully characterized. In order to
successfully colonize the mammalian host and triatomine vector, T. cruzi assumes three
distinct morphological forms at different developmental stages (Fig. 1) (13). Amastigote
and epimastigote forms replicate by binary fission in mammalian cells and the hindgut
of the triatomine vector, respectively. Trypomastigote forms are nonreplicative and are
present at two distinct life cycle stages: (i) in the bloodstream of the mammalian host
(bloodstream-form trypomastigotes) and (ii) in the rectum and feces of vectors (infec-
tive metacyclic trypomastigotes).
Infective metacyclic trypomastigotes are deposited on the skin of the mammalian
host in fecal droplets extruded by a blood-feeding triatomine bug. Parasites enter
through the bite site, skin abrasions, or mucosa, such as the conjunctiva. This mecha-
nism, via the vector feces rather than mouthparts, is known as stercorarian transmis-
sion. Once internalized, motile trypomastigotes invade nucleated cells via both lysosome-
dependent and -independent mechanisms (reviewed in references 14 and 15). The
parasite is then taken up into a membrane-bound (parasitophorous) vacuole, which
subsequently fuses with a lysosome; exposure to decreasing pH stimulates parasite
differentiation to the intracellular amastigote form and its concomitant release into the
cytosol over a period of 4 to 5 days. Here, amastigotes multiply asexually to form
pseudocysts, which can arise in a variety of host tissues but predominantly do so in
cardiac, smooth, and skeletal muscles and reticuloendothelial cells in the liver, spleen,
and lymphatic system. Within pseudocysts, amastigotes differentiate into trypomastig-
otes that, upon cell lysis, can either infect adjacent tissues to initiate new replicative
cycles or disseminate throughout the bloodstream and lymph. Without antitrypano-
somal treatment, infection persists for the duration of the mammalian host’s life.
Triatomine bugs feeding on an infected host may ingest extracellular trypomastig-
otes, which pass to the midgut where transformation to an intermediate spheromas-
tigote form occurs. Differentiation of spheromastigotes into epimastigotes occurs in
response to decreasing environmental glucose levels as the blood meal is digested (13).
Epimastigotes multiply by binary fission in the hindgut and migrate to the rectum,
where they attach hydrophobically to the waxy gut cuticle by their flagella and transform
into infective metacyclic trypomastigotes, thus completing the life cycle.
Routes of Transmission
Vector-borne transmission. Vector-borne transmission remains the predominant
route of new human infections in regions of endemicity. Historically, vector-borne
transmission has occurred in ecologically determined areas throughout continental
Latin America, from Mexico to the northern 50 to 60% of the territories of Argentina
and Chile (16). Infected vectors and reservoir animals are not infrequent in the southern

half of the continental United States, but vector-borne transmission to humans is rarely
detected (reviewed in multiple sections that follow).
Congenital transmission. Reported vertical transmission rates are variable, ranging
from 0% in some studies to more than 15%; the pooled transmission risk in a recent
meta-analysis was 4.7% (17). Factors associated with a higher risk include younger
maternal age (reflecting more recent infection), maternal immunological responses,
higher maternal parasitemia, twin births, and HIV coinfection (18–21). Infected infants
are detected regularly in screening programs in Spain and sporadically in other countries
with Latin American immigrant populations (22–24).
Blood-borne transmission. In the early 1990s, T. cruzi infection was found in 1 to
60% of donated blood units in Latin American blood banks (25). Since then, blood
donation screening has been established as a major component of Chagas disease control
programs (26). With the addition of Mexico in 2012, screening of blood components for T.
cruzi is now required in all countries of endemicity in Latin America, and reported donor
prevalence has decreased markedly (26, 27).
Organ-derived transmission. Transplantation of an organ from a T. cruzi-infected
donor can transmit T. cruzi to the recipient, but the risk varies by organ type. In cohorts
of kidney recipients from infected donors in the United States and Argentina, trans-
mission occurred in 13% and 19%, respectively (28, 29). The transmission rate among
10 U.S. liver transplant recipients was 20% (28). The risk from heart transplant in the
same U.S. series was 75% (3 of 4); use of the heart from an infected donor is contraindicated
(28, 30).
Oral transmission. Outbreaks of acute T. cruzi infection due to contaminated fruit or
sugar cane juice have been reported in several countries of Latin America (31, 32). Most
case clusters are small, affecting family groups in the Amazon and attributed to fruits like
açaí (33). The largest reported outbreak was associated with a 10% attack rate among
students and staff at a school in Caracas; home-pressed guava juice was implicated (34).
EPIDEMIOLOGY AND ECOLOGY

Global Burden of Chagas Disease
The Chagas disease control initiatives instituted throughout Latin America since
1991 constitute a major public health success story. Thanks to vector control programs,
blood bank screening, and in some countries, congenital Chagas disease screening
programs, global estimates have decreased from 18 million in 1991 to less than 6
million infected individuals in 2010 (Table 1) (3, 35). Incidence estimates have fallen
from 500,000 in 1991 to 30,000 new T. cruzi infections per year in 2010 (3). As vectorial
transmission has come under increasing control, the proportion attributable to other
routes has grown: currently, 22.5% of incident infections are estimated to occur
through congenital transmission, and in some areas, oral transmission may be more
frequent than the traditional vector-borne route (3, 31).
Triatomine Vector Biology

More than 130 triatomine species have been reported in the Western Hemisphere,
many known to carry T. cruzi (16, 36). However, a few species are disproportionately
responsible for T. cruzi transmission to humans, due to their propensity to colonize
human houses and/or the peridomestic environment. These include Triatoma infestans
and Panstrongylus megistus in the Southern Cone (Argentina, Bolivia, Brazil, Chile,
Paraguay, and Uruguay), Triatoma dimidiata in southern Mexico and Central America,
and Rhodnius prolixus in Central America and northern South America (16). These
species have been the major targets of the regional control initiatives. The elimination
of R. prolixus from Central America and the near elimination of domestic T. infestans
from much of the Southern Cone are responsible for the steep decline in new infections
and very low prevalence in children throughout most of the historic zone of endemicity
(37, 38). However, in the Gran Chaco, an ecological zone that straddles southern Bolivia,
northeastern Argentina, and parts of Paraguay, the prevalence of house infestation and
transmission remains very high (39, 40).

TABLE 1 Countries in which Chagas disease is endemic, estimates of national

seroprevalence, and numbers of infected inhabitantsa
Estimated T. cruzi infection
prevalenceb
Region Country % prevalence No. of infections
North America United States NDA 240,000 to 350,000c
Mexico 0.779 876,458
Central America Belize 0.330 1,040

Costa Rica 0.170 7,667
El Salvador 1.298 90,222
Guatemala 1.230 166,667
Honduras 0.918 73,333
Nicaragua 0.523 29,300
Panama 0.515 18,337
South America Argentina 3.641 1,505,235

Bolivia 6.104 607,186
Brazil 0.606 1,156,821
Chile 0.700 119,660
Colombia 0.956 437,960
Ecuador 1.380 199,872
Guyana, French Guiana, & Suriname 0.839 12,600
Paraguay 2.130 184,669
Peru 0.440 127,282
Uruguay 0.238 7,852
Venezuela 0.710 193,339
Total 1.056 5,742,167d

aVector-borne T. cruzi transmission occurs or occurred until recently in parts of these countries.
bDisease burden estimates are for the year 2010 and are based on references 3 and 7. NDA, no data
available.
cThe figure for the United States reflects the estimated number of infected immigrants from countries of
disease endemicity of Latin America. No estimate of locally acquired infections is currently available.
dExcluding the United States.
The domestic environment is rich in blood meal sources, both human and animal.
Crevices in adobe walls and dark spaces within animal corrals and poultry nests provide
safe diurnal refuges for triatomines. Rhodnius species, which nest in palm crowns in the
sylvatic environment, can infest thatch roofs. Triatomines of both sexes require at least
one blood meal during each of the five nymphal stages, and females need a blood meal
to lay eggs. Thus, both male and female nymphs and adults may carry T. cruzi, with
infection rates increasing with age. Only adults have wings. Most domestic triatomine
species feed nocturnally and complete their blood meals without waking the host (36).
The major Latin American vectors defecate during or immediately after taking a blood
meal (41). Many sylvatic triatomine species colonize the nests of their blood meal
sources and are found in close association with specific rodent or marsupial species (16,
36). Sylvatic triatomine adults may be attracted by light to invade human dwellings,
which can lead to sporadic human infections (42, 43). Some triatomine species, such as
T. dimidiata, can infest both domestic and sylvatic sites (44).
Triatomine Distribution in the United States

Eleven triatomine species have been reported in the United States: Triatoma ger-
staeckeri, T. incrassata, T. indictiva, T. lecticularia, T. neotomae, T. protracta, T. recurva, T.
rubida, T. rubrofasciata, T. sanguisuga, and Paratriatoma hirsuta (Fig. 2 and Table 2) (9,
36). Triatomines are present from coast to coast across the southern two-thirds of the
continental United States (Fig. 3). In field collections, vectors are often found in specific
microenvironments (woodpiles, rock piles, rodent nests, livestock pens, and dog ken-
nels) (9). Natural T. cruzi infections have been documented in all species except the
rarely collected T. incrassata and P. hirsuta (9, 45).
The two species with the widest geographic distribution in the United States are T.
sanguisuga and T. protracta. The former has been reported from Texas to the Atlantic

FIG 2 Photographs of U.S. triatomine species of the genera Triatoma and Paratriatoma. Image size relative to the scale bar represents the average length of
each species. The Triatoma incrassata photo is courtesy of E. Barrera Vargas, the T. recurva and Paratriatoma hirsuta photos are courtesy of R. Hoey-Chamberlain
and C. Weirauch, and the T. protracta protracta photo is courtesy of G. Lawrence (DPDM/CDC). All other images are from reference 9 (photos by S. Kjos).
coast and as far north as Illinois, the latter from Texas to California (9, 46). A recent
review by the Wheeling-Ohio County West Virginia Health Department turned up 10
specimens of T. sanguisuga archived since 1969 and adds this state (long assumed to
have the vector) to the confirmed list (46). T. sanguisuga was also recently reported in
Delaware (47). T. protracta has been extensively collected in association with its favored
blood meal hosts, the woodrats (Neotoma spp.); the prominent above-ground nests of
these rodents make sylvatic collection relatively straightforward for this species (9, 48).
T. protracta includes three morphologically distinct subspecies in the United States, T.
TABLE 2 Triatomine vectors in the United Statesa

Frequency of
Species collectionb Rangeb Ecological association(s) T. cruzi prevalence
T. sanguisuga Frequent AL, AR, DE, FL, GA, IL, IN, KS, KY, LA, Highly diverse, including woodrats, Moderate prevalence,
MD, MO, MS, NC, NJ, OH, OK, PA, other rodents, armadillos, opossums, very widespread
SC, TN, TX, VA, WV dogs, chickens, horses; frequent in
peridomestic settings; invades houses
T. gerstaeckeri Very frequent Eastern NM, central TX Sylvatic and peridomestic settings, dog High prevalence,
kennels, rodent burrows; frequently especially in dog
invades houses kennel collections
T. protracta Frequent AZ, CA, CO, NM, NV, west TX, UT Close association with woodrats Moderate prevalence,
(Neotoma spp.); attracted by lights widespread
T. rubida Frequent AZ, southern CA, NM, southwest TX Woodrat nests, disturbed environments Usually low, but focal
in AZ and Mexico; reports of house collections with
colonization in Sonora, Mexico high prevalence
T. lecticularia Infrequent FL, GA, MO, NM, OK, SC, TN, TX, UTc Houses, dog kennels, woodrat nests in Can be high in
TX; peridomestic settings collections from
woodrat nests
T. indictiva Infrequent AZ, NM, TX Found in woodrat nests and near lights Moderate in sparse
data
T. recurva Infrequent Southern half of AZ Associated with rodents, especially rock Low to moderate
squirrels
T. neotomae Rare TX Found in woodrat nests Can be high in
collections from
woodrat nests
T. incrassata Rare Southern AZ Unknown No naturally infected
specimen reported
P. hirsuta Rare CA, AZ, NV Found in woodrat nests, near lights, and No naturally infected
invading houses specimen reported
T. rubrofasciata Rare Jacksonville, FL; Honolulu, HI Roof rats (Rattus rattus); found in houses 2 infected bugs
in FL and HI, chicken coops in HI reported in HI
aBased on our review of literature from 1939 to 2011 (9) plus new data in references 46, 47, and 335.
bFrequency and range are based on published reports; absence of reports from a given area often reflects lack of field research rather than true absence of vectors.
Ranges of all species except T. rubrofasciata extend into Mexico.
cSeveral other states are listed for T. lenticularia in reference 45 and reproduced by reference 278 but were not confirmed in our 2011 review (9). We follow the
approach advocated by Ryckman (49), in which reports prior to Usinger (336) are treated with caution in the absence of later verification. Utah was added based on
the recent report in reference 335.

FIG 3 Ranges of the four most frequently identified triatomine species in the continental United States.
Based on references provided in Table 2.
protracta protracta in California, Nevada, Utah, Arizona, and New Mexico, T. protracta
woodi in Texas, and T. protracta navajoensis in the Four Corners area, where Colorado,
Utah, Arizona, and New Mexico meet (49). Thousands of specimens of T. sanguisuga and
T. protracta have been reported in literature dating back to the 1930s, and these species
were found in or near the residences of humans with locally acquired T. cruzi infection
in Tennessee, Louisiana, Mississippi (T. sanguisuga), and California (T. protracta) (9,
50–53). In field collections, both species frequently have T. cruzi infection, with rates
generally in the 15 to 30% range (9, 48).
T. gerstaeckeri has a more limited range, encompassing south-central Texas and
southeastern New Mexico, but is one of the most frequently collected species, perhaps
in part because of its propensity to infest dog kennels and other peridomestic struc-
tures (54). T. gerstaeckeri constituted more than 70% of several thousand vectors
submitted through a citizen science project based at Texas A&M University (55).
Collections of T. gerstaeckeri show high rates of T. cruzi infection, often ⬎60% (9, 55).
Infected T. gerstaeckeri insects were collected in the house of a child with acute T. cruzi
infection in south Texas in 2006 (56).
Texas and the southwestern states have the highest triatomine species diversity,
with at least seven species in Texas and six in Arizona (Table 3) (9, 54). A spatial analysis
of bugs submitted through the Texas citizen science initiative showed geographic
overlap among species but with T. gerstaeckeri predominantly in south-central Texas, T.
sanguisuga in the eastern portion, T. rubida in west Texas, and T. indictiva in a small area
of central Texas (54). T. gerstaeckeri reports showed earlier seasonality than T. san-
guisuga, possibly because of the earlier arrival of high temperatures in the southern
part of the state. Like all passive surveillance, there may be reporting biases in these
data. The authors observe that they received few submissions from west Texas (and
perhaps for this reason, few T. protracta specimens). They attribute this to lower human
population density and/or less effective outreach (54), but lower rates of Internet access
in rural counties could also play a role.

TABLE 3 Triatomine vectors and Trypanosoma cruzi-infected mammals by state in published reports
T. cruzi infection identified in:
State Vector(s) reported Vector(s) Wildlife Dogs
AL T. sanguisuga Yes Raccoon, opossum
AR T. sanguisuga
AZ T. protracta, T. rubida, T. indictiva, Yes Raccoon, ringtail, skunk, woodrats, other rodents
T. recurva, T. incrassata, P. hirsuta
CA T. protracta, T. rubida, P. hirsuta Yes Skunk, woodrats, other rodents Yes
CO T. protracta
DE T. sanguisuga
FL T. sanguisuga, T. lecticularia, T. rubrofasciata Yes Raccoon, opossum, skunk, gray fox
GA T. sanguisuga, T. lecticularia Yes Raccoon, opossum, skunk, gray fox, bobcat, Yes
coyote, feral swine
HI T. rubrofasciata Yes
IL T. sanguisuga
IN T. sanguisuga Yes
KS T. sanguisuga Yes
KY T. sanguisuga Raccoon, opossum
LA T. sanguisuga Yes Opossum, nine-banded armadillo Yes
MD T. sanguisuga Raccoon, opossum
MO T. sanguisuga, T. lecticularia Yes Raccoon
MS T. sanguisuga Yes
NC T. sanguisuga Raccoon, opossum
NJ T. sanguisuga
NM T. lecticularia, T. protracta, T. gerstaeckeri, Yes Woodrats, other rodents
T. rubida, T. indictiva
NV T. protracta, P. hirsuta
OH T. sanguisuga
OK T. sanguisuga, T. lecticularia Yes Raccoon, opossum Yes
PA T. sanguisuga
SC T. sanguisuga, T. lecticularia Gray fox Yes
TN T. sanguisuga, T. lecticularia Yes Raccoon Yes
TX T. sanguisuga, T. lecticularia, T. protracta, Yes Raccoon, opossum, nine-banded armadillo, Yes
T. gerstaeckeri, T. rubida, T. indictiva, skunk, American badger, coyote, woodrats,
T. neotomae other rodents, bat
UT T. protracta, T. lecticularia
VA T. sanguisuga Yes Raccoon, opossum, coyote Yes
WV T. sanguisuga
The ranges of all United States species extend into Mexico, with the exception of T.
rubrofasciata (49, 57). T. rubrofasciata is associated with rats and is thought to have
been carried from North America globally on sailing ships in the 18th century (58). In
the United States, this species has been reported in Jacksonville, Florida, and Honolulu,
Hawaii, consistent with its predominant distribution in ports.
Allergic Reactions to Triatomine Antigens

T. gerstaeckeri, T. protracta, T. recurva, T. rubida, and T. sanguisuga have been
implicated in allergic reactions in the United States (59). Such reactions are due to
vector salivary antigens, not the infection status of the vector. Most reactions consist of
a pruritic welt where the bite occurred. Severe reactions may involve angioedema,
urticaria, dyspnea, gastrointestinal symptoms, and/or anaphylaxis (59). Severe reactions
may necessitate treatment with epinephrine (60). Reports are most frequent in Arizona
and California; the most commonly identified species are T. protracta and T. rubida, and
the most frequent scenario is house invasion by an adult triatomine (59). In a study in
southern California, allergic reactions consistent with those provoked by triatomine
exposure were reported by 13% of residents of desert areas with frequent triatomine
sightings, compared to 4% of those living in suburban Los Angeles County (61).
Wild and Domestic Animal Reservoirs

Wildlife reservoirs. Trypanosoma cruzi infection has been reported in more than 150
species of mammals from eight orders, and it is widely believed that all mammals are
susceptible (62). Birds and cold-blooded vertebrates are refractory to infection (63). The

epidemiological importance of particular species is highly variable, depending on local

ecology and parasite transmission dynamics. Maintenance reservoirs have persistent
infection, while amplifier reservoirs are those that display characteristics that favor
transmission, such as high parasitemia levels (64). As with humans, most infected animals
are chronically infected, and therefore, detection may be reliant on a combination of
examination of peripheral blood smears, culture isolation, serological testing, and PCR;
both relative ease of trapping and variations in the performance of diagnostic assays
contribute to bias in reported prevalence levels. Across the range of endemicity,
Dasypus novemcinctus (nine-banded armadillo) and Didelphis species (opossums) are
prominent sylvatic reservoirs and amplifiers of infection. Trypanosoma cruzi is able to
infect almost all tissues in its mammalian hosts, including atypical sites like the cornea
of Thrichomys apereoides (spiny rat) (65) and the anal scent glands of Didelphis species
(66), enabling the latter to function as both host and vector. In addition to vector-borne
transmission, many sylvatic mammals are prone to alternate transmission routes,
including oral infection via ingestion of infected vectors, congenital infection, and
exposure to contaminated bodily secretions (67). These biological features may pre-
dispose such hosts to infection with multiple strains, due to high transmission intensity
and efficiency (68, 69).
In the United States, T. cruzi infection has been demonstrated in more than 24
wildlife species, including raccoons, opossums, armadillos, foxes, mice, squirrels, coy-
otes, skunks, and wood rats (9). Recent studies have expanded this list to include
additional rodent (70, 71), bat (72), and deer species (73). Reported seroprevalence rates
fluctuate quite widely within species, ranging from 15 to 90% in raccoons (70, 74, 75),
9 to 100% in skunks (70, 76), 8 to 33% in opossums (76, 77), and 20 to 76% in woodrats
and other rodents (70, 78–80). The prevalence varies depending on ecology and local
diversity and density of vector species and, in some cases, between sexes, with female
denning activities being associated with increased triatomine contact (76, 81). High
infection rates in some mammals, such as wood rats and raccoons, may result from
frequent insectivory (82). Experimental infection studies and the high attack rates in
human outbreaks of orally transmitted Chagas disease suggest that ingestion of
infected vectors or vector fecal material is a very efficient transmission route (34, 82). In
contrast, consumption of raw T. cruzi-infected meat did not result in experimental
infection in one study (82).
Canine Chagas disease. Dogs are important in peridomestic cycles in Latin America,
both as vector blood meal sources and T. cruzi infection reservoirs (83, 84). In the Chaco
region of Argentina where the disease is hyperendemic, dogs have been shown to be
highly infective to vectors and are thought to be a key reservoir sustaining transmission
to humans (85). In the United States, T. cruzi-infected dogs have been reported from
Tennessee, South Carolina, Georgia, Virginia, Louisiana, California, Oklahoma, and Texas
(reviewed in reference 9). Infected dogs may develop acute and chronic manifestations
similar to those in humans, including acute myocarditis, arrhythmias, chronic dilated
cardiomyopathy, congestive heart failure, and sudden death (86). Several recent sur-
veys in Texas demonstrate widespread canine T. cruzi infection, especially in working
dogs and those living in kennels (87–90). The prevalence in these surveys varied widely.
Some studies demonstrated significant discordance between diagnostic tests and a
substantial number of dogs whose infection status was unresolved with the testing
performed (89). The highest infection rate, 71% by serology, was reported in the
investigation of a Texas kennel where several dogs suffered sudden death suspected to
be due to acute Chagas disease (87). Triatomines collected in dog kennels and near
houses in Texas show high prevalences of canine blood meals and T. cruzi infection (87,
91), suggesting that dogs may be an important peridomestic host.
Transmission Potential in the United States

With the exception of the rarely collected species T. neotomae, T. incrassata, and
P. hirsuta, all U.S. vector species have been reported to invade human dwellings (54, 60).
A total of 2,883 specimens of 7 different vector species were submitted to the Texas

citizen science project (54). Of these, 17% were collected inside human dwellings; the
highest proportions were for T. rubida and T. protracta. Houses with refuges like
woodpiles, rock piles, and brush and those with structural gaps through which vectors
can pass are more vulnerable to vector invasion (50, 60, 92). Rarely, the presence of T.
protracta or T. recurva nymphs has been reported inside houses, suggesting possible
colonization (60). Window screens, air conditioning, and caulking of gaps in house
construction may be protective against such invasion (60).
Detection of human blood meals is frequent in tested triatomines, especially those
collected in and around human dwellings and in other spaces where humans congre-
gate. In a recent study in Texas, human blood was detected in 59% (30/51) of T.
gerstaeckeri bugs; the second most frequent blood meal was canine (17/51, 33%),
followed by more than a dozen other vertebrate hosts (93). In this study, collection sites
were largely domestic or peridomestic, and human blood meals were found in 77%
(17/22) of T. gerstaeckeri bugs collected inside homes; mixed blood meals were fre-
quent. In another study from Texas, vectors were collected in dog kennels and woodrat
nests, as well as domestic settings (91). In this study, dogs (10/33, 30%) were the
predominant blood meal source for T. gerstaeckeri, followed by woodrats (Neotoma
micropus) (7/33, 21%); human blood was identified in a single bug. All 40 T. protracta
bugs were collected in woodrat nests and had fed exclusively from Neotoma micropus
(91). In a Louisiana study conducted near the house of the 2006 autochthonous human
infection, 43 T. sanguisuga bugs were collected; 53% had fed from American green tree
frogs, 49% from humans, and 30% from raccoons; detection of blood from multiple
host species was frequent (94). In the Arizona-Sonora Desert Zoo, human blood was
detected in all 7 T. rubida bugs tested; 5 of 7 had other blood meal sources detected,
including pig, sheep or goat, dog, mouse, rat, or woodrat (95). Human blood was also
detected in 2 of 3 T. recurva bugs collected elsewhere in southern Arizona (95).
The coincidence of human blood meals and T. cruzi infection in triatomines has been
described as indicating the “potential for Chagas disease” in the United States (93–95).
Clearly, transmission to humans occurs; more investigation is needed to quantify the
risk, since most infections likely go undetected. However, the small number of locally
acquired T. cruzi infections detected in humans stands in contrast to the moderate to
high T. cruzi prevalence rates in dogs, raccoons, opossums, and woodrats. Compared to
major South American vectors, such as R. prolixus and T. infestans, North American
vectors appear to have somewhat longer time intervals from blood meal to defecation
and may be less likely to defecate on the host (96–99). Vectors rarely colonize houses
in the United States, and well-constructed houses with window screens provide effec-
tive barriers against domestic invasion. Perhaps most importantly, stercorarian trans-
mission is inefficient; mathematical models based on data from the Gran Chaco, where
transmission to humans is the highest in the world, estimate that a single human T.
cruzi infection requires on average 900 to 4,000 contacts with infected vectors (69).
MOLECULAR EPIDEMIOLOGY
Trypanosoma cruzi Genotypes
Trypanosoma cruzi is a highly genetically diverse parasite, estimated to have di-
verged from its most recent common ancestor 3 to 4 million years ago (100). Scientific
consensus currently defines a minimum of six genetic lineages, or discrete typing units
(DTUs [TcI to TcVI]) (101), plus a potential seventh, bat-associated genotype (TcBat),
most closely related to TcI (102–105). Multiple molecular markers confirm a largely
clonal population structure, which maintains the identity of major DTUs, interspersed
with recombination events (106). TcI through TcIV form monophyletic clades, while TcV
and TcVI resulted from recent hybridization of TcII and TcIII (100, 107). Genomic data
support this evolutionary model. TcI to TcIV display substantial allelic homozygosity
resulting from long-term, recurrent, and dispersed gene conversion, whereas TcV and
TcVI have natural heterozygosity and minimal distinction, with shared intact alleles
from their parental DTUs (100, 107–111).

Each T. cruzi DTU is characterized by distinct but often overlapping transmission

ecologies (112). TcI, TcII, TcV, and TcVI are commonly associated with domestic cycles
and are the genotypes found in most human infections. Investigators have long
observed that gastrointestinal Chagas disease is more frequent in the Southern Cone
than further north in Latin America and hypothesized a connection to different
circulating T. cruzi strains (113). However, there remains no clear, unequivocal evidence
of influence of particular lineages on the progression or clinical outcome of human
Chagas disease (reviewed in reference 114). Domestic TcI is distributed from the
Amazon Basin northwards and is the principal DTU found in humans in Venezuela,
Ecuador, and Colombia (115–117). TcI also circulates in arboreal ecotopes between
Didelphis species and the triatomine tribe Rhodniini (118, 119), with secondary cycles
among rodents and sylvatic Triatoma species in highland valleys in Bolivia, Peru, and
Chile (120–123). Sylvatic TcI populations are characterized by high levels of genetic
diversity (124–130), while human infections are associated with divergent, more ge-
netically homogenous strains (128, 131–133). In contrast, TcII, TcV, and TcVI appear less
variable overall (100) and are predominant in domestic cycles in the Southern Cone
(112, 134, 135). However, recent whole-genome sequencing of clinical TcII isolates has
revealed more extensive intra-DTU diversity than previously reported (110). Sylvatic
reservoirs of TcII, TcV, and TcVI are less well delineated than for TcI, but TcII has been
increasingly detected in Brazilian primates (130, 136, 137). In addition, TcV and TcVI
have been demonstrated in domestic dogs from Argentina to as far north as Colombia
(107, 138–141). TcIII is transmitted by Panstrongylus geniculatus to D. novemcinctus and
other burrowing mammals in terrestrial transmission cycles from the Amazon Basin to
Argentina (142–144). The known host range of this DTU has expanded to include dogs,
grisons, and foxes in Brazil (145). TcIV, perhaps the most neglected DTU, circulates
sympatrically with TcI in wild primates in the Amazon (146) and raccoons and dogs in
the United States (147). TcIV can invade the domestic environment in Venezuela (113,
116) and has been isolated from patients during oral-infection outbreaks in the Brazilian
Amazon (146, 148–151) and Colombia (152). Finally, TcBat has been isolated from
Chiroptera species across Brazil (102), Panama (103), Colombia (105), and Ecuador (104)
and is potentially infective to humans (153).
Trypanosoma cruzi Molecular Epidemiology in the United States

The majority of genotyping activities have concentrated on vectors and reservoir
hosts. In an extensive analysis of U.S. vectors and mammalian hosts, all five autoch-
thonous human cases were reported as TcI (online supplement in reference 119). In a
more recent series of presumed-autochthonous chronic T. cruzi infections in Texas
blood donors, the authors were limited by genetic marker resolution and therefore
unable to distinguish among TcII, TcV, and TcVI (154).
To date, TcI and TcIV are the only DTUs detected among the six triatomine species
examined, with no absolute associations between parasite genotype and vector (Table
4). Higher proportions of TcIV have usually been identified in T. sanguisuga, T.
indictiva, and T. lenticularia, compared to a predominance of TcI in T. gerstaeckeri,
T. protracta, and T. rubida (78, 87, 89, 119, 155–157). However, except for studies of
vectors collected by the Texas citizen science initiative (156, 157), samples sizes
were far too small to make any meaningful extrapolations. The observation of
potential TcII/V/VI autochthonous human infections in Texas is noteworthy but
challenging to interpret without clear evidence of these genotypes circulating in
local vector species (154). Similarly, a study of T. protracta collected in California
encountered issues distinguishing among TcII/V/VI and was unable to establish the
presence of infections with these lineages (158). Further investigations are war-
ranted to confirm the presence of these DTUs in the United States, using a larger
panel of more highly resolutive markers, in conjunction with phylogenetic analyses
incorporating all representative T. cruzi DTUs; neither of these studies examined
parasite sequence homology to TcI (154, 158).

TABLE 4 Trypanosoma cruzi genotypes reported in triatomine vectors in the United States
No. (%)
No. (%) that were:
Total no. T. cruzi No.
Location Vector examined positive typed TcI TcIV TcI/IV Genotyping methoda Reference
TX T. gerstaeckeri 16 16 (100) 16 10 (63) 4 (25) 2 (13) SL-IR; TcSC5D gene and 87
SNPs in subset
TX T. gerstaeckeri 897 574 (64) 548 294 (54) 189 (34) 65 (12) TcSC5D; SL-IR on subset 156
South TX T. gerstaeckeri 18 9 (50) 9 6 (67) 1 (11) 2 (22) SL-IR 89
TX T. gerstaeckeri 11 1 (9) NR 100% NR SL-IR 157
TX T. gerstaeckeri NR NR 3 2 (67) 0 1 (33) SL-IR, 24S ␣-subunit rRNA, 119
18S rRNA
TX T. gerstaeckeri 19 13 (100) 13 13 (100) 0 0 18S rRNA sequencing 71
TX T. gerstaeckeri 1 1 (100) 0 0 SL-IR 337
TX T. indictiva 67 32 (48) 28 9 (32) 17 (61) 2 (7) TcSC5D; SL-IR on subset 156
TX T. lenticularia 66 44 (67) 42 9 (21) 25 (60) 8 (19) TcSC5D; SL-IR on subset 156
TX T. lenticularia 2 2 (100) 2 2 (100) 18S rRNA sequencing 71
TX T. protracta 19 3 (16) 2 2 (100) 0 0 TcSC5D; SL-IR on subset 156
Southwestb T. protracta 14 1 (7) 1 1 (100) 0 0 TcSC5D; SL-IR on subset 156
Northern CA T. protracta 29 16 (55) 13 13 (100) 0 0 RFLP (HPS60, GPI), SL-IR, 48
24S ␣-subunit rRNA,
sequencing of Rb19, TR,
and COII-ND1
Southern CA T. protracta 68 21 (31) 9 7 (78) 2 (22) 0 RFLP (HPS60, GPI), SL-IR, 48
24S ␣-subunit rRNA,
sequencing of Rb19, TR
and COII-ND1
Southern CA T. protracta 161 34 2c 0 0 0 24S ␣-subunit RNA 158
(21.1)
TX T. protracta 9 4 (44) NR 100% NR SL-IR 157
TX T. rubida 64 11 (17) 7 6 (86) 1 (14) 0 TcSC5D; SL-IR on subset 156
Southwestd T. rubida 40 7 (18) 5 5 (100) 0 0 TcSC5D; SL-IR on subset 156
South TX T. rubida 2 0 0 0 0 0 SL-IR 89
TX T. rubida 299 69 (23) NR 100% NR SL-IR 157
West TX T. rubida 39 24 (62) 24 24 (100) 0 0 TcSC5D 155
TX T. sanguisuga 20 13 (65) 13 2 (15) 9 (69) 2 (15) SL-IR; TcSC5D and SNPs in 87
subset
TX T. sanguisuga 315 158 (50) 135 21 (16) 107 (79) 7 (5) TcSC5D; SL-IR on subset 156
Southeaste T. sanguisuga 45 12 (27) 12 2 (17) 10 (83) 0 TcSC5D; SL-IR on subset 156
Midwestf T. sanguisuga 7 4 (57) 3 0 3 (100) 0 TcSC5D; SL-IR on subset 156
FL, GA T. sanguisuga 4 4 (100) 0 0 SL-IR, 24S ␣-subunit rRNA, 119
18S rRNA
LA T. sanguisuga 12 8 (67) 6 6 (100) 0 0 SL-IR, 24S ␣-subunit rRNA, 78
18S rRNA
aSL-IR, spliced-leader intergenic region; SNPs, single-nucleotide polymorphisms; RFLP, restriction fragment length polymorphism.
bAZ, CA, NM.
cTyped as II/VI.
dAZ, NM.
eAL, FL, GA, KY, LA, NC, TN, VA.
fIN, KS, MO, OH, OK.
Among reservoir hosts, TcI and TcIV are the principal DTUs identified in United
States (Table 5). Similar to vector surveys, sample sizes are insufficient to reveal any
strict correlations between host and parasite genotype; current data demonstrate both
lineages circulating among mammalian hosts in variable proportions. Finally, a few
studies in Louisiana reported rodents harboring TcII, alongside other mixed TcI, TcIV,
and TcVI infections (78). Additional sampling efforts will be necessary to delineate the
frequency and ecology of TcII/V/VI in the United States.
Issues Underlying T. cruzi Genotyping Data Collection

To accurately interpret T. cruzi genotypic data, biological and logistical limitations
relating to both parasite infection dynamics and genotyping methodologies must
be considered. Trypanosoma cruzi genotyping can be conducted on clinical samples
(blood or tissue) or parasite axenic cultures, obtained through hemoculturing or
xenodiagnosis. Due to low levels of peripheral parasitemia, especially in chronically

TABLE 5 Trypanosoma cruzi genotypes reported in mammalian hosts in the United States
No. (%) of samples typed
Other DTUs
as:
Location(s) (no. Total no. reported (no. of
of individuals) Host genotyped TcI TcIV TcI/IV samples) Genotyping method Reference
CA (2), LA (1), Humans 5 5 (100) 0 0 SL-IR, 24S ␣-subunit 119
TX (2) rRNA, 18S rRNA
TX Humans 6 0 0 0 TcII-V-VI (4), TcI/ PCR-RFLP (SL-IR, 24S 154
TcII-V-VI (2)a ␣-subunit rRNA, 18S
rRNA), sequencing
CA (1), OK (1), Canis lupus familiaris 7 0 6 (86) 1 (14) SL-IR, 24S ␣-subunit 119
SC (2), TN (1), (domestic dog) rRNA, 18S rRNA
Unknown (2)
TX C. lupus familiaris 2 1 (50) 0 1 (50) SL-IR 89
(domestic dog)
TX C. lupus familiaris 15 9 (60) 5 (33) 1 (7) SL-IR, sequencing of 87
(domestic dog) TcSC5D
TX C. lupus familiaris 4 4 (100) 0 0 SL-IR 337
(domestic dog)
TX C. lupus familiaris 6 5 (83) 1 (17) 0 SL-IR, 24S ␣-subunit 88
(domestic dog) rRNA, 18S rRNA, COII
FL (16), GA Procyon lotor 64 2 (3) 61 (95) 1 (2) SL-IR, 24S ␣-subunit 119
(45), MD (1), (raccoon) rRNA, 18S rRNA
TN (1), SC (1)
GA P. lotor (raccoon) 5 0 5 (100) 0 SL-IR, confirmatory 70
sequencing
TX P. lotor (raccoon) 11 10 (91) 0 1 (9) TcSC5D 75
TX P. lotor (raccoon) 2 0 2 (100) 0 SL-IR, 24S ␣-subunit 338
rRNA, 18S rRNA, COII
IL P. lotor (raccoon) 5 0 5 (100) 0 SL-IR, 24S ␣-subunit 339
rRNA, confirmatory
sequencing
KY P. lotor (raccoon) 2 0 2 (100) 0 SL-IR, 24S ␣-subunit 339
rRNA, confirmatory
sequencing
MO P. lotor (raccoon) 1 0 1 (100) 0 SL-IR, 24S ␣-subunit 339
rRNA, confirmatory
sequencing
GA Lemur catta (ring- 3 0 3 (100) 0 SL-IR, 24S ␣-subunit 119
tailed lemur) rRNA, 18S rRNA
GA (1), Macaca mulatta 2 1 (50) 0 1 (50) SL-IR, 24S ␣-subunit 119
unknown (1) (rhesus macaque) rRNA, 18S rRNA
Tx M. mulatta (rhesus 33 18 (55) 13 (39) 2 (6) SL-IR, 24S ␣-subunit 338
macaque) rRNA, 18S rRNA, COII
AL (1), FL (6), Didelphis virginiana 15 15 0 0 SL-IR, 24S ␣-subunit 119
GA (6), LA (2) (opossum) (100) rRNA, 18S rRNA
TX D. virginiana 4 4 0 0 SL-IR, 24S ␣-subunit 338
(opossum) rRNA, 18S rRNA, COII
GA (1), LA (2) Dasypus 3 2 (67) 1 (33) 0 SL-IR, 24S ␣-subunit 119
novemcinctus rRNA, 18S rRNA
(nine-banded
armadillo)
GA Mephitis mephitis 1 0 1 (100) 0 SL-IR, 24S ␣-subunit 119
(striped skunk) rRNA, 18S rRNA
GA M. mephitis (striped 4 1 (25) 3 (75) 0 SL-IR, confirmatory 70
skunk) sequencing
TX M. mephitis (striped 2 1 (50) 1 (50) 0 SL-IR, 24S ␣-subunit 338
skunk) rRNA, 18S rRNA, COII
TX Neotoma micropus 23 10 (43) 13 (57) 0 SL-IR, confirmatory 70
(Southern plains sequencing
woodrat)
TX N. micropus 1 1 (100) 0 0 18S rRNA sequencing 71
(Southern plains
woodrat)
(Continued on next page)

TABLE 5 (Continued)
No. (%) of samples typed
Other DTUs
as:
Location(s) (no. Total no. reported (no.
of individuals) Host genotyped TcI TcIV TcI/IV of samples) Genotyping method Reference
GA Sigmodon hispidus 2 0 2 (100) 0 SL-IR, confirmatory 70
(hispid cotton rat) sequencing
GA Otospermophilus 1 0 1 (100) 0 SL-IR, confirmatory 70
variegatus (rock sequencing
squirrel)
TX Peromyscus leucopus 3 3 (100) 0 0 18S rRNA sequencing 71
(white-footed
mouse)
TX Chaetodipus hispidus 1 1 (100) 0 0 18S rRNA sequencing 71
(hispid pocket
mouse)
TX S. hispidus (hispid 1 1 (100) 0 0 18S rRNA sequencing 71
cotton rat)
TX Baiomys taylori 1 1 (100) 0 0 18S rRNA sequencing 71
(northern pygmy
mouse)
TX Liomys irroratus 1 1 (100) 0 0 18S rRNA sequencing 71
(Mexican spiny
pocket mouse)
LA Peromyscus 20 16 (80) 0 0 TcII (2), TcI/TcII SL-IR, 24S ␣-subunit 78
gossypinus and (1), TcII/TcIV rRNA, 18S rRNA
Mus musculus (1)
(mouse spp.)
LA Neotoma floridana 3 2 (67) 0 0 TcII/TcIV (1) SL-IR, 24S ␣-subunit 78
rRNA, 18S rRNA
TX Nycticeius humeralis 1 1 (100) 0 0 SL-IR, 24S ␣-subunit 72
(evening bat) rRNA, 18S rRNA, COII
aThe authors were unable to distinguish between non-TcI DTUs.
infected patients, direct genotyping is insensitive but may be improved if multiple

specimens are tested (159). The principal limitation of parasite isolation is selection bias
for specific clones, due to higher growth rates or culture conditions to begin with
(160–162) and, subsequently, by loss of diversity from long-term maintenance in axenic
culture or animals (163–167). Hemoculture recovery rates are usually less than 30%
among chronic patients (168) and are dependent upon parasite load and distribution
in the initial inoculum. Xenodiagnosis can generally recover more parasite strains, but
biases may result from variable vector permissibility to specific strains (169–171). Further-
more, circulating clones isolated by hemoculture or xenodiagnosis may be different
from those sequestered in tissues due to differential strain tropisms (172–174) and can
vary even between sequential blood samples (175). Similar biases affect sylvatic T. cruzi
sampling, with certain reservoir species more heavily represented in survey data due to
their relative ease of capture and the presence of detectable parasitemia.
The most commonly used genotyping techniques for clinical and field specimens
involve analysis of size polymorphisms in multicopy genetic markers, particularly the
nuclear spliced-leader intergenic region (SL-IR), 24␣ rRNA, and 18S ribosomal DNA
(rDNA) (Tables 4 and 5) (176, 177). The major confounder associated with the use of any
multicopy gene is the level of intraclone copy number and undefined chromosomal
orthology, which can prevent direct comparability between strains. The SL-IR is present
in many hundreds of copies per parasite genome; the copies are not necessarily
identical and may instead comprise a predominant haplotype accompanied by a low
abundance of minor paralogous sequence types more closely related in identity to
other DTUs, likely resulting from their shared evolution (110, 178, 179). In this scenario,
it is virtually impossible to distinguish between a monoclonal DTU infection containing
multiple divergent SL-IR sequences and a polyclonal infection consisting of different
major DTU parasites. This issue is minimized when using conventional PCR, as generally,

the most common gene sequence is amplified in a reaction. However, recently, a

number of deep sequencing studies reported results based on sequencing millions of
copies of the SL-IR locus that seem to indicate the occurrence of almost all DTUs in
infected rodents and primates in the United States (180, 181). Parallel deep sequencing
of appropriate biologically cloned controls to exclude low-abundance haplotypes has
thus far yielded equivocal evidence; further investigations are essential to define the
applicability of this technique to characterize natural multiclonal infections (182, 183).
CLINICAL MANIFESTATIONS
Acute T. cruzi Infection
The acute phase begins 1 to 2 weeks after vector-borne transmission and lasts approx-
imately 8 weeks. Patients are most commonly asymptomatic or experience mild, nonspe-
cific symptoms such as fever. A T. cruzi abscess or chagoma may occur at the site of
inoculation. Parasite entry via the conjunctiva may result in unilateral eyelid swelling
(the Romaña sign) (184). However, eyelid swelling can be caused by an allergic reaction
to triatomine salivary or fecal antigens; confirmed diagnosis of T. cruzi infection is
obligatory, even in the setting of vector exposure and an apparent Romaña sign. Severe
acute Chagas disease, including myocarditis, pericardial effusion, and/or meningoen-
cephalitis, is rare, but when it occurs, the mortality risk is high (5, 185). In the absence
of the Romaña sign or severe manifestations, individual infections are seldom diag-
nosed during the acute phase.
Orally transmitted T. cruzi infection has been reported to cause more severe acute
morbidity and higher mortality than vector-borne infection (186, 187). Microepidemics
appear to be fairly frequent in the Amazon basin, due to sylvatic vectors contaminating
produce, such as açaì or sugarcane (31). In the Caracas outbreak, mentioned above, 103
people were infected, of whom 59% had electrocardiogram (ECG) abnormalities and
20% were admitted to hospital, and one person died from acute Chagas myocarditis
(32, 34). Alterations in T. cruzi surface glycoproteins caused by exposure to gastric acid
may increase parasite invasiveness, providing a possible explanation for the increased
severity of orally acquired Chagas disease (188, 189).
Congenital Chagas disease is acute infection in the newborn. Most infected infants
are asymptomatic or have mild findings, but a small percentage present with severe
disease or die in utero (18, 190). Manifestations may include low birth weight, prema-
turity, low Apgar scores, hepatosplenomegaly, anemia, and thrombocytopenia (18,
190). Severely affected neonates may have meningoencephalitis, gastrointestinal me-
gasyndromes, myocarditis, pneumonitis, and/or respiratory distress (18). Women who
receive antitrypanosomal therapy prior to conception are significantly less likely to
transmit T. cruzi to their infants (23, 191).
Chronic T. cruzi Infection

One to 2 months after infection, parasitemia falls below levels detectable by
microscopy, and the patient passes into the chronic phase of T. cruzi infection (2, 5).
Chronic T. cruzi infection without signs or symptoms of Chagas disease is designated
the indeterminate form (2, 5, 192). Over a period of years to decades, an estimated 20
to 30% of infected individuals develop cardiomyopathy (2, 5). A retrospective cohort
analysis of Brazilian blood donors estimated progression to cardiomyopathy to occur at
a rate of 1.85% per year (193). Chagas cardiomyopathy features chronic inflammation
in all chambers and damage to the conduction system and cardiac muscle (194). The
most frequent early signs are right bundle branch block or left anterior fascicular block
and segmental left ventricular wall motion abnormalities (185, 194, 195). Later mani-
festations appear decades after infection and include ventricular arrhythmias, sinus
node dysfunction and bradycardia, persistent or intermittent complete heart block, an
apical aneurysm usually in the left ventricle, thromboembolic phenomena, and pro-
gressive dilated cardiomyopathy. Patients may experience palpitations, syncope, and
systemic and pulmonary emboli, with high risk of sudden death or death from progressive
heart failure. (185, 194, 195).

TABLE 6 Chagas disease diagnostic testing by clinical context

Clinical scenario Testing modality(ies), specimen type(s), and schedule (reference[s])
Suspected chronic T. cruzi infection
All persons (symptomatic or asymptomatic) with IgG serologyb by two distinct assays, preferably based on different
epidemiological risk factorsa; high priority to screen antigens (16)
children and women of child-bearing age, especially
if pregnant or planning pregnancy
Persons at risk for acute T. cruzi infection

Suspected contact with infected vector PCR (microscopy)c in blood between 2 and 8 wks postexposure, IgG
serology at 6–8 wks
Infant of T. cruzi-infected mother PCR (microscopy)c in blood at birth and 1–3 mos; IgG serology at
9–12 mos (216)
Recipient of blood components, organ, or tissue from Serial PCR in blood 1⫻/wk in mos 1–2, 1⫻/2 wks in mos 3–4,
infected donor 1⫻/mo in mos 5–6, then based on clinical scenario (30)
Laboratory accident Serial PCR (microscopy)c in blood 1⫻/wk for 6–8 wks, IgG serology at
6 to 8 wks (340)
Persons at risk for T. cruzi reactivation

Prospective organ or tissue recipient with risk factors IgG serology by two distinct assays (341, 342)
Transplant recipient with chronic T. cruzi infection Serial quantitative PCR in bloodd 1⫻/wk in mos 1–2, 1⫻/2 wks in
mos 3–4, 1⫻/mo in mos 5–6, then based on clinical scenario (203);
for heart transplant patients, histology in endomyocardial biopsy
specimen, especially in setting of suspected rejection
HIV-T. cruzi-coinfected patient with signs of reactivation PCRd/microscopy in tissue, blood, CSF as clinically indicated
T. cruzi-infected patient with iatrogenic PCRd/microscopy in tissue, blood, CSF as clinically indicated
immunosuppression (chemotherapy, corticosteroids)
and signs of reactivation
aEpidemiological risk factors for T. cruzi infection include birth or residence or maternal birth or residence in a country with endemic vectorial transmission (see Table
1); residence in areas of the United States with high rates of vector-human contact, especially if the patient reports triatomine bites and/or house invasion.
bPlasma is not an approved biospecimen for some FDA-cleared tests; serum is acceptable for all FDA-cleared tests.
cPCR is substantially more sensitive than microscopy in peripheral blood.
dPositive PCR in blood does not diagnose reactivation; rising parasite load in blood is generally the first indication. Positive PCR in cerebrospinal fluid (CSF) indicates
reactivation.
Gastrointestinal involvement is much less frequent than cardiomyopathy. Esopha-

geal manifestations range from asymptomatic motility disorders through mild achalasia
to megaesophagus (196). Patients may experience dysphagia, odynophagia, esopha-
geal reflux, weight loss, aspiration, and regurgitation. Patients with colonic involvement
may have prolonged constipation, fecaloma, volvulus, bowel ischemia, or megacolon.
Symptomatic gastrointestinal disease, like symptomatic cardiac disease, usually appears
several decades after infection.
Chagas Disease in the Immunocompromised Host

Organ-derived infection. Acute T. cruzi infection in organ transplantation recipients
may lead to a relatively severe clinical spectrum, with manifestations that include acute
myocarditis and congestive heart failure (197). In recent years, as screening of donors
has become more frequent, most donor-derived infections have been detected by PCR
monitoring prior to symptom onset, allowing prompt antitrypanosomal treatment and
favorable outcomes (28). Current recommendations suggest monitoring the recipient
of an organ from an infected donor for at least 6 months, at which point the frequency
can be decreased (Table 6) (30).
Reactivation in cardiac transplant recipients. Cardiac transplantation is an ac-
cepted treatment for end-stage Chagas cardiomyopathy (198, 199). In a large Brazilian
cohort, survival of patients transplanted for Chagas cardiomyopathy was better than
among those with idiopathic or ischemic cardiomyopathy and T. cruzi reactivation was
a rare cause of death (200, 201). Data from a smaller cohort of patients transplanted for
end-stage Chagas cardiomyopathy in the United States also demonstrated survival
similar to that among patients transplanted for other etiologies (202). The most common
manifestations of reactivation are fever and acute myocarditis in the transplanted heart.
Patients may also develop inflammatory panniculitis and cutaneous nodules (197).

Central nervous system (CNS) involvement occurs infrequently. All patients with dilated
cardiomyopathy and a history of significant residence in continental Latin America should
be screened (203). For those found to be infected, posttransplant monitoring should
include histopathology of the explanted heart and subsequent endomyocardial biopsy
specimens and serial peripheral blood monitoring by quantitative PCR (Table 6) (203).
Reactivation of Chagas disease in HIV-coinfected patients. The most common
clinical manifestation of T. cruzi reactivation in HIV-coinfected patients is meningoen-
cephalitis with or without a mass lesion (204). The case fatality rate for CNS reactivation
is very high. The presentation is often confused with CNS toxoplasmosis (205, 206); T.
cruzi should be considered in the differential diagnosis of CNS mass lesions in HIV-
infected patients (207, 208). Acute reactivated myocarditis is another frequent mani-
festation and may be obscured by preexisting chronic cardiomyopathy (209). New
arrhythmias or conduction system abnormalities, pericardial effusions, or cardiac de-
compensation should prompt testing for reactivation. Subcutaneous nodules resem-
bling erythema nodosum and parasitic invasion of the peritoneum, stomach, or
intestine can occur but are uncommon (210). Five cases of T. cruzi reactivation in
HIV-infected Latin American immigrants have been reported in the United States since
1992; all presented as CNS syndromes and were treated initially as toxoplasmosis (205,
206, 211–213).
DIAGNOSTIC TECHNIQUES
The choice of modality to diagnose Chagas disease is determined by the clinical
setting and suspected phase of infection. In general, techniques to directly detect the
parasite are used in the acute phase and suspected reactivation, whereas IgG serology
is the mainstay of diagnosis in the chronic phase (Table 6).
Microscopy
In acute, congenital, or reactivated infection, trypomastigotes may be detectable by
light microscopy in thick and thin smears from whole blood or buffy coat with routine
staining (e.g., Giemsa stain) (214). When acute or reactivation meningoencephalitis is
suspected, cerebrospinal fluid samples should be concentrated by the thin-layer cell
preparation technique, stained, and examined by light microscopy. Microscopy is useful
due to fast turnaround time, wide availability, and high specificity, but its sensitivity is
lower than that of molecular techniques (215, 216).
Molecular Techniques
The most sensitive primer sequences originate from satellite or kinetoplast
minicircle DNA (217–219). A recent publication from the CDC outlines an algorithm that
incorporates testing by multiple primer sets in a quantitative assay to optimize perfor-
mance and reliability (218). Several recent initiatives have addressed standardization of
qualitative and quantitative PCR for clinical use (217, 220). In acute or early congenital
infection, PCR has substantially higher sensitivity than microscopy and is the diagnostic
test of choice (190, 219). PCR results are variably positive in chronic T. cruzi infection,
depending on specimen volume, primers, DNA extraction methods, and level of
experience of the laboratory (220). Blood clot or buffy coat preparations may provide
higher sensitivity than whole blood, but these preparations may not be widely available
in routine clinical laboratories (218, 221). PCR has recently been utilized in several
clinical trials as an early indicator of treatment failure; use in this setting requires
rigorous standardization and criteria for patient inclusion (for example, positive results
by PCR in at least one of 3 pretrial 10-ml specimens) (222, 223). In chronically infected
patients at risk because of immunosuppression, a rise in parasite load by quantitative
PCR in serial specimens is the earliest indicator of reactivation, enabling treatment
before onset of symptoms (224, 225).
Diagnostic Serology
Diagnosis in the chronic phase of Chagas disease relies on detection of host IgG
against T. cruzi antigens (16). Currently, the main methods in use are enzyme-linked

immunosorbent assays (ELISAs), immunofluorescence assays (IFAs), and immunochro-

matographic strip or cassette tests. Confirmed diagnosis requires positive results by at
least two assays, preferably based on different antigens (for example, parasite lysate
and recombinant antigens) (16). The sensitivities and specificities of the available assays
are not sufficient for a single assay to be used alone for diagnosis, especially in a setting
where prevalence is low and pretest probability is not high. The IgG trypomastigote
excreted-secreted antigen immunoblot (TESA-blot) is used as a confirmatory test in
blood banks and clinical practice in Brazil (226, 227). Preparation of the test strips using
antigen from trypomastigotes in cell culture requires specialized infrastructure and may
be prone to interbatch and laboratory variability. The banding pattern may differ by T.
cruzi DTU, suggesting different antigenic characteristics between strains (228). Conven-
tional serology in cord and infant blood reflects transferred maternal IgG until around
9 months of age.
IgM-based assays have been evaluated for the diagnosis of acute T. cruzi infection,
with a special focus on use for congenitally infected infants in settings where molecular
assays are not available (226). IgM TESA-blot showed sensitivity of 58% compared to a
consensus definition of infection and 80% compared to PCR in congenitally infected
infants in Bolivia; in these two analyses, the specificities were 98% and 94%, respec-
tively (190, 229). The specificity of IgM assays utilizing whole T. cruzi lysate was ⬍30%
in a similar population of congenitally infected infants (230). In the United States, PCR
is the assay of choice for the diagnosis of acute and congenital T. cruzi infection (216).
HCT/P
Serological screening is recommended for donors of human cells, tissues, and
tissue-based products (HCT/P) with epidemiological risk factors; for example, those who
were born or lived in areas of endemicity or whose mothers were born in such areas.
Two assays are approved by the U.S. Food and Drug Administration (FDA) for living and
cadaveric donor screening, Ortho ELISA (Ortho-Clinical Diagnostics, Inc., Raritan, NJ)
and Abbott PRISM (Abbott Diagnostics, Abbott Park, IL) (231). These tests are currently
available only in blood donor testing laboratories. The Organ Procurement and Trans-
plantation Network/United Network for Organ Sharing (OPTN/UNOS) Disease Transmis-
sion Advisory Committee also recommends the use of an FDA-cleared diagnostic ELISA
to test living donors (232, 233). For living donors, a positive screening test should
prompt referral for appropriate diagnostic testing and clinical evaluation (192).
Histopathology
Trypanosoma cruzi causes tissue damage through cellular lysis, inflammatory re-
sponse, and fibrotic replacement (234). The spectrum of histopathology related to T.
cruzi infection has been the subject of several recent reviews (235–239). The most
important target organ is the heart, where chronic pathology includes multichamber
damage, most prominent in the ventricles and often severe enough to form an apical
aneurysm (235, 236). The spectrum of microscopic pathology includes myofiber de-
generation, interstitial fibrosis, and patchy inflammation predominantly comprised of
lymphocytes, macrophages, plasma cells, and eosinophils; neutrophils are not com-
monly observed. The patterns of inflammation and fibrosis can be focal or diffuse
throughout the layers of the myocardium. Fibrotic plaques may be observed on the
epicardium (235, 236). Intracellular amastigote pseudocysts are rarely observed in
chronic pathology, especially with limited tissue sampling, but demonstrable parasite
persistence appears to be associated with higher-grade inflammation in the chronic
phase (236, 240, 241). Histopathology can play an important diagnostic role in the
setting of suspected reactivation. Careful examination of endomyocardial biopsy spec-
imens for nests of intracellular parasites can help distinguish rejection from T. cruzi
reactivation in cardiac transplant recipients (203, 242). In immunosuppressed patients
with suspected skin manifestations of T. cruzi reactivation, histopathology may reveal
the parasite and confirm the diagnosis (238). The diagnosis of T. cruzi reactivation was

made on a brain biopsy specimen in a patient with HIV and cerebral lesions of unknown
etiology (205).
ETIOLOGICAL TREATMENT AND CLINICAL MANAGEMENT

Antitrypanosomal Drugs
Benznidazole and nifurtimox are the only drugs with proven efficacy against Chagas
disease (2). Benznidazole is considered the first-line treatment, because of better
tolerance and more comprehensive efficacy data (1, 243). Benznidazole is a prodrug
that requires metabolism by parasite enzymes to become active; metabolites appear to
act through multiple mechanisms to interrupt T. cruzi glutathione and trypanothione
pathways (243). Dermatologic side effects are frequent and consist of rashes and
photosensitization (244, 245). Dermatitis occurs with significantly higher frequency in
females than males (244). Most rashes are mild and can be managed with antihista-
mines or topical steroids without interrupting treatment (246). Treatment should be
suspended immediately for severe or exfoliative dermatitis or dermatitis associated
with fever and lymphadenopathy. Peripheral neuropathy is dose dependent, usually
occurs late in the course of therapy, and should prompt immediate treatment inter-
ruption; resolution may take months. Bone marrow suppression is rare and requires
immediate interruption of treatment. Clinical and laboratory monitoring for side effects
should occur regularly throughout the course of treatment. Benznidazole tolerance is
substantially better in children than in adults and in children younger than 7 years than
in older children (247). This better safety profile correlates directly with more rapid
elimination of the drug in younger age groups (248).
Benznidazole was approved by the FDA in August 2017 (249) and became com-
mercially available in the United States as of 14 May 2018. The drug is marketed in the
United States by Exeltis, a U.S.-based division of Insud Pharma (previously called Chemo
Group) (250). The approval covers treatment of T. cruzi infection in children 2 to
12 years of age (250); usage for other age groups is off-label. As of 2019, prescriptions
require submission of a completed order form to Exeltis FastAccess (https://www
.benznidazoletablets.com/en/Treatment). Urgent requests for benznidazole should be
made by telephone (877-303-7181).
Nifurtimox, a nitrofuran, impedes T. cruzi carbohydrate metabolism through the
inhibition of pyruvic acid synthesis. The most common side effects are anorexia and
weight loss, experienced by up to 70% of patients. Other frequent adverse effects
include nausea, vomiting, irritability, and insomnia (251, 252). Rarely, patients develop
peripheral neuropathy, usually manifested as paresthesias. The peripheral neuropathy
is dose dependent, appears late in the course of therapy, and requires cessation of the
drug. Nifurtimox is better tolerated by children than adults. As of 2019, nifurtimox is not
approved by the FDA but is provided by the CDC under investigational protocols
(https://www.cdc.gov/parasites/chagas/health_professionals/tx.html; telephone no.,
404-718-4745).
Several new drug candidates (posaconazole and the ravuconazole prodrug E1224)
have been tested in recent trials, but so far, none has shown acceptable efficacy (222,
223). All of the participants in these trials were from the Southern Cone. Although the
posaconazole trial was carried out in Spain, 75 of the 78 subjects acquired their
infections in Bolivia (223). Recent reviews have called for the inclusion of patients from
diverse locations within Latin America, representing all of the major T. cruzi strains that
infect humans (253). A novel aspect of these trials was the use of carefully standardized
PCR assays to document treatment failure (254). Of those treated with posaconazole, 80
to 90% had detectable parasitemia by 12 months posttreatment, compared to ben-
znidazole failure rates of 6% (per protocol) to 38% (intention to treat) (223). Similar
results were demonstrated in a Bolivian trial of E1224, a related drug (222). These trials
demonstrated that, with rigorous standardization, PCR may be useful as an early
indicator of treatment failure, at least in populations of patients with infection acquired
in the Southern Cone.

Acute and Congenital T. cruzi Infection

Acute infection has been an absolute indication for treatment since the drugs first
became available in the 1970s (255). In acute and early congenital T. cruzi infection,
antitrypanosomal therapy reduces the severity of symptoms, shortens the clinical
course, and decreases the duration of detectable parasitemia (255, 256). In severe acute
disease, treatment can be lifesaving. Cure rates in acute and congenital infection are
estimated at 80 to 99% (255–258).
Treatment of Chronic T. cruzi Infection

Evaluation of antitrypanosomal drug efficacy in chronic T. cruzi infection is challeng-
ing. PCR, while a potential indicator of treatment failure, is not a true test of cure, since
many persons with chronic T. cruzi infection will have circulating parasite levels below
the threshold of detection of the assay. Conventional IgG serology is considered the
only sensitive indicator of infection, but it requires years to decades to revert to
negative after successful treatment (259). The longer the duration of infection, the more
durable the antibody response, with women treated after age 15 taking a median of
27 years to revert to negative serology (191). Age is often used as a proxy for infection
duration, since in communities where the disease is endemic, most infections are
acquired in childhood. Testing using experimental lytic antibody assays shows conver-
sion to negative results more quickly than with conventional serology but still requires
years, even in children (260). In the 1990s, two placebo-controlled trials of benznidazole
treatment in children with chronic T. cruzi infection showed approximately 60% cure
rates based on lytic antibody assays 3 to 4 years after treatment (261, 262). These
studies made early diagnosis and antitrypanosomal drug therapy the standard of care
for children and prompted the establishment of large-scale pediatric screening pro-
grams in high-prevalence locations (16, 263).
Treatment of chronic infection in adults remains a topic of debate (264, 265). The
fundamental question is whether antitrypanosomal therapy decreases the risk that an
infected person will develop cardiac morbidity from T. cruzi. Observational data pub-
lished in 2006 suggested that benznidazole treatment significantly decreased the
progression of Chagas cardiomyopathy in adults (266). Since progression only occurs in
20 to 30% of those with infection and takes decades to become clinically evident, the
ideal trial would require large study populations followed for 20 years or more, a
virtually impossible clinical trial design. The design of the BENEFIT trial (Benznidazole
Evaluation for Interrupting Trypanosomiasis; ClinicalTrials.gov registration no.
NCT00123916), a randomized, double-blinded, placebo-controlled trial, was based on
the observation that patients who already have cardiac morbidity are more likely to
have further progression than those with normal cardiac status (267). Eligible patients
were required to have cardiac findings consistent with established Chagas cardiomy-
opathy, and the primary outcome consisted of any of the following: death, resuscitated
cardiac arrest, sustained ventricular tachycardia, insertion of a pacemaker or implant-
able cardioverter-defibrillator, cardiac transplantation, new heart failure, or other throm-
boembolic event. To the disappointment of many in the Chagas disease community, the
trial showed no significant difference for the primary composite outcome, despite
significantly higher conversion to negative PCR results in the treatment group com-
pared to the results for the placebo group (268).
The patient populations in the observational and trial populations differed substan-
tially. The nonrandomized study subjects had a mean age of 39, and two-thirds had
normal cardiac function at baseline (266). In contrast, the BENEFIT trial population had
a mean age of 55, all had cardiac damage based on electrocardiographic abnormalities,
and nearly half had decreased ejection fraction at baseline, indicating ventricular
dysfunction (268). The question of whether treatment provides clinical benefit for those
with no or very early cardiac signs therefore remains unanswered (269). The only clear
take-away messages are that the younger the patient the higher the probability of
benefit and that active screening is essential to identify infected individuals before they
become symptomatic. As in earlier publications (192), treatment recommendations

TABLE 7 Recommendations for antitrypanosomal drug treatment according to Chagas disease phase and form, patient age, and clinical
status
Recommendation on antitrypanosomal drug treatment by Chagas Strength of recommendation;
disease phase, form, and demographic group quality of evidencea
Should always be offered
Acute T. cruzi infection (including congenital infection in 1st mo of life) Strong; moderate
Children ⱕ12 yrs old with chronic T. cruzi infection Strong; high
Children 13–18 yrs old with chronic T. cruzi infection Strong; low
Reactivated T. cruzi infection in immunosuppressed patient Strong; moderate
Reproductive-age women planning future pregnancies Strong; moderate
May be offered with consideration of potential risks and benefits,

uncertainties, and patient preferences
Adults with normal ECG and cardiac function Discretionary; weak
Adults with early signs of cardiomyopathy Discretionary; weak
Recommendation against treatment

During pregnancy Strong; weak
During lactation Weak; weak
Patients with advanced cardiomyopathy Strong; moderate
Patients with gastrointestinal Chagas disease that impairs absorption Weak; weak
aBasedon the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system (343). The GRADE system offers only two grades of
recommendations: “strong” and “weak” or “discretionary.” Strong recommendations are provided when the balance of desirable versus undesirable effects is clear.
Weak or discretionary recommendations require an assessment of the evidence and decision making based on a consideration of potential risks and benefits,
uncertainties, and patient preferences (344).
remain stratified by age and clinical status and require balancing the risk of adverse
effects with the probability and uncertainty of benefit (Table 7).
Management of the Immunocompromised Host

In organ transplant recipients with reactivation, a standard course of benznidazole
or nifurtimox is effective in ameliorating clinical symptoms and shortening the duration
of microscopically detectable parasitemia. Prior treatment or posttransplant prophy-
laxis has not been shown to decrease the risk of reactivation; posttransplant prophy-
laxis is not generally administered in heart transplant centers in Latin America (270). As
no reliable test of cure exists, treated patients are considered to remain at risk for
reactivation (225). Organ recipients at risk of reactivation should have regular moni-
toring of blood by quantitative PCR, with treatment based on demonstration of rising
parasite load in blood (Table 6) (225, 271). T. cruzi reactivation should be included in the
differential diagnosis of febrile episodes and apparent rejection crises, and endomyo-
cardial biopsy specimens should be examined for evidence of T. cruzi myocarditis in the
transplanted heart. Reactivation in an HIV-coinfected patient is treated with standard
courses of antitrypanosomal treatment and optimization of antiretroviral therapy (272).
The utility of and optimal regimen for secondary prophylaxis are unknown.
HUMAN CHAGAS DISEASE IN THE UNITED STATES

Disease Burden among Latin American Immigrants
No population-representative data exist to make an unbiased estimate of T. cruzi
infection prevalence in the United States. Several studies of T. cruzi seroprevalence in
convenience samples of Latin American immigrants have been conducted. In Los
Angeles, 59 (1.24%) of 4,755 Latin-American-born residents had confirmed T. cruzi
infection (273). The prevalence was higher among participants older than 40 than in
those 18 to 40 (1.42% versus 0.95%), and it was higher among immigrants from El
Salvador than those from Mexico (3.45% versus 0.79%) (273). A community health
clinic-based program to screen Latin American immigrants in East Boston reported an
overall prevalence of 0.87% (19/2,183), with prevalence rising with age (0/101 [0%] for
age ⬍20, 10/1,562 [0.64%] for age 20 to 39, and 9/507 [1.78%] among those 40 years
or older) (274).
Based on the reported number of immigrants from countries of Chagas disease
endemicity of Latin America and estimated national T. cruzi seroprevalence in their

countries of origin, there were an estimated 240,000 to 350,000 infected persons in the
United States in 2010; the upper end of the range includes an estimate for undocu-
mented immigrants, whereas the lower end does not (7). All estimates of Chagas
disease burden in the United States have major uncertainties, and the method used for
these estimates carries several potential biases. The demographics of Latin American
immigrants in the United States may not reflect those of the general population in their
countries of origin, and their significant exposure risk ended when they left their home
countries years earlier (275). Chagas disease prevalence is highly heterogeneous in
countries of endemicity; depending on geographic sources of immigrants, the preva-
lence in immigrants could be either higher or lower than the national average. For
example, in Spain, Bolivian immigrants appear to have a higher prevalence of Chagas
disease than the estimated national prevalence, possibly because they are more likely
to come from departments with high prevalence, such as Cochabamba and Santa Cruz,
than from departments with low prevalence, such as Oruro or Potosí (3, 8). Similar
systematic information for Mexican and Central American immigrants in the United
States is lacking, although data from Los Angeles support the notion that infection
prevalence is higher among immigrants from some Mexican states than others (273).
Finally, the composition of migrant populations entering the United States has changed
in recent years, with a higher proportion of families and children from Central America
than in earlier migrations, in which adult men from Mexico predominated (275).
National T. cruzi prevalences are higher in El Salvador, Guatemala, and Honduras than
in Mexico (3), but the younger age of migrants would have the effect of decreasing the
likely prevalence in migrants compared to that in earlier waves of older migrants. The
success of vector control programs has dramatically decreased the prevalence of T. cruzi
infection among children in Latin America over the past 30 years, and the limited data
from Boston suggest that pediatric Chagas disease is infrequent in Latin American
immigrants in the United States (274).
Autochthonous Vector-Borne Transmission to Humans

Available data indicate that autochthonous vector-borne T. cruzi transmission to
humans is rare in the United States (276, 277). A longitudinal study in repeat blood
donors yielded a point estimate of zero incidence, with an upper 95% confidence limit
of 0.61 per million (277). House colonization is rare, and vector-human contact occurs
primarily in peridomestic areas, when vectors invade houses, or when humans spend
time in sylvatic sites with enzootic T. cruzi transmission (9, 278).
Prior to initiation of blood bank screening in 2007, all reported vector-borne
infections in the United States were detected because of acute symptoms and/or
the presence of a vector in the vicinity of the case (Table 8). Four infections were
reported in Texas and one each in California, Tennessee, and Louisiana. Five of
seven cases occurred in infants or small children, and six were in the acute phase
at the time of detection. In retrospect, one of the Texas infections, reported to be
in a 2- to 3-week old infant with no other details provided, may actually have been
congenital (279). In California, a contemporaneous survey demonstrated positive
complement fixation results in 6 (2.5%) of 241 residents of the community associ-
ated with the 1982 case compared to 1 (0.2%) of 637 persons surveyed in a major
urban area (53).
Since 2007, blood bank screening has resulted in the publication of an additional 35
putative autochthonous T. cruzi infections (50, 280–285). All were in the chronic phase
and detected on serological screening by blood centers. States postulated to be
sources of infection include Mississippi, Texas, Louisiana, Arizona, and California, but
with the exception of the CDC investigation (50), all were either individual case reports
or focused on a single state, raising the likelihood of sampling bias. The CDC investi-
gation estimated that locally acquired T. cruzi infection was likely to account for
between 5.5% and 7.5% of confirmed positive blood donations (50). Unlike earlier case
reports, all of these putative autochthonous infections were in adults in the chronic
phase; thus, the location and timing of transmission events are unknown. Some reports

TABLE 8 Case data for reported autochthonous vector-borne Trypanosoma cruzi infections in the United States
Evidence of autochthonous vector-
State of borne origin; putative state of
residence Age Sex Diagnostic modality Phase Yr detected Yr infected acquisition Reference(s)
TX 10 mos F Microscopy of Acute 1955 1955 Peridomestic infestation; TX 345
peripheral blood
TX 2–3 wks M Not reported Acute 1955 1955 No details provided—perhaps 279
congenital, given reported age; TX
CA 56 yrs F Microscopy of Acute 1982 1982 Adult uninfected T. protracta in 53, 346
peripheral blood house; CA
TX 7 mos M Histology of cardiac Acute 1983 1983 No vectors found but search made 347
tissue in winter, house in poor
condition; TX
TN 18 mos M T. cruzi PCR in Acute 1998 1998 T. cruzi-infected T sanguisuga found 52
peripheral blood in child’s crib; TN
TX 12 mos M Microscopy of Acute 2006 2006 Mother uninfected, T. cruzi-infected 56
pericardial fluid T. gerstaeckeri near house; TX
LA 74 yrs F Serology and Chronic 2006 Unknown T. sanguisuga infestation, 10/18 51
hemoculture positive by T. cruzi PCR; LA
MS 44 yrs M Blood donor screening Chronic 2007 Unknown T. sanguisuga found on property, 50
also extensive hunting of known
T. cruzi reservoir species; MS
NRa NR NR 14 cases detected on Chronic 2006–2010 Unknown Blood donors not from Latin 50
blood donor America and not primarily Spanish
screening speaking
TX 59 yrs M Blood donor screening Chronic 2007 Unknown Rural TX, including deer hunting in a 282
place with infected T. gerstaeckeri
TX 69 yrs M Blood donor screening Chronic 2007 Unknown Rural TX, some travel to Mexico 282
TX 47 yrs F Blood donor screening Chronic 2007 Unknown Residence in rural TX and LA 282
TX 72 yrs M Blood donor screening Chronic 2010 Unknown Residence in rural TX 282
TX 21 yrs M Blood donor screening Chronic 2011 Unknown Extensive camping in TX and MO 282
TX 83 yrs M Blood donor screening Chronic NR Unknown Former/current residence considered 283
high risk; TX
TX 61 yrs F Blood donor screening Chronic NR Unknown Former/current residence considered 283
high risk; TX
TX 71 yrs M Blood donor screening Chronic NR Unknown Occupation considered high risk; TX 283
TX NR NR Blood donor screening Chronic NR Unknown Camping considered likely risk; TX 283
high risk; TX
high risk; TX
high risk; TX
TX 52 yrs M Blood donor screening Chronic NR Unknown Current residence, occupation, 283
hunting considered moderate risk;
TX
high risk; TX
high risk; TX
high risk; TX
CA 19 yrs M Blood donor screening Chronic 2009 Unknown Lack of international travel, 285
extensive camping history; TX
TX 28 yrs M Blood donor screening Chronic 2014 Unknown Reported vectors near childhood 284
home in AZ; TX resident at time
of detection
AZ 16 yrs F Blood donor screening Chronic NR Unknown T. cruzi-infected T. rubida found near 280
home; AZ
aNR, not reported.
speculate on potential sources of triatomine contact, including hiking, camping, hunt-

ing, and peridomestic woodpiles and brush (50, 282, 283, 285). In other cases, infected
individuals had exposure in multiple states with known sylvatic cycles and no hypoth-
eses could be formed as to which was the most likely site of acquisition (280, 282).

TABLE 9 Published reports of transfusion-related Trypanosoma cruzi transmission in the United States
Yr of Implicated blood component,
transmission State Recipient characteristics donor origin Reference
1988 NY 11-yr-old female with Hodgkin lymphoma, developed fever Platelets, Bolivia 348
and pericarditis, trypomastigotes seen on blood smear;
treated with nifurtimox and recovered
1988 CA 17-yr-old male post-bone marrow transplant with Not specified, Mexico 349
fulminant acute Chagas disease
1989 TX 59-yr-old female with metastatic colon cancer on Unknown; had received ⬎500 350
chemotherapy, granulocytopenic, disseminated units, including RBCs,
intravascular coagulation; developed fever, pulmonary platelets
infiltrates, bradycardia and atrioventricular block;
parasites seen on bone marrow aspirate; died within
36 h of diagnosis
1997 FL 60-yr-old female with multiple myeloma; T. cruzi-infected Platelets, Chile 351
donor unit detected during research study; recipient
asymptomatic, treated with nifurtimox; died of
underlying disease several yrs later
2002 RI 3-yr-old female with stage 4 neuroblastoma on Platelets, Bolivia 352
chemotherapy, neutropenic, fever, trypomastigotes seen
on blood smear; treated with nifurtimox but died of her
underlying disease
2004 NY 64-yr-old male with non-Hodgkin lymphoma and Platelets, Argentina 287
chemotherapy-induced thrombocytopenia; T. cruzi found
on serological testing during look-back study
2006 NY 62-yr-old male; T. cruzi found on serological testing during Platelets, Argentina 287
look-back study
Blood Donor Screening and Transfusion Transmission

Prior to the institution of blood donor screening, five transfusion-associated T. cruzi
infections were documented in the United States, two in 1988, one in 1989, one in 1997,
and one in 2002 (Table 9) (9, 286). Evaluation of the recipients of blood components
from infected donors identified two additional transmission events in 2004 and 2006,
from separate donations from the same donor (287). Most infected recipients had
underlying malignancies and were immunosuppressed (9, 287). Donors from the
Southern Cone were implicated in 5 of the 6 cases where the source was known. In two
cases, the blood component was unknown; in all others, the implicated units were
platelets. Based on tracing of 350 recipients of blood components from infected donors, the
risk associated with a platelet unit was estimated to be 13.3% (95% confidence interval [CI],
5.6 to 25.7), compared to zero for packed red blood cells (RBCs) (95% CI, 0 to 0.15) and
frozen plasma/cryoprecipitate (95% CI, 0 to 3.7) (286). Several of the acutely infected
recipients had severe manifestations of Chagas disease, including acute myocarditis,
acute atrioventricular block, severe congestive heart failure, pericarditis with T. cruzi in
the pericardial fluid, and possible meningoencephalitis.
Blood donation screening began in January 2007 using the Ortho ELISA, which had
been licensed by FDA the previous month, with the radioimmunoprecipitation assay
(RIPA) as the confirmatory test (288). The FDA licensed the Abbott PRISM chemilumi-
nescent immunoassay (ChLIA) as a screening test in 2010 and the Abbott enzyme strip
assay (ESA) as a supplemental test in 2011. Guidance from the FDA in 2010 recom-
mended screening of all donations, regardless of previous screening results (277).
Universal screening enabled an analysis of results from two or more serial specimens
from 4.22 million repeat donors representing 6.06 million person-years of follow-up
(277, 290). No incident T. cruzi infections were detected, corresponding to zero autoch-
thonous transmission incidence, with an upper 95% confidence limit of 0.61 incident
cases per million person-years (277). In 2017, final FDA guidance endorsed a one-time
screening approach, recommended against the use of donor questions to assess risk
based on low sensitivity, and required further testing of screen-positive donations with
the Abbott ESA (291, 292). Screening was estimated to cover 75 to 90% of the U.S.
blood supply as of 2008 (293).

Several ancillary studies were conducted during the early years of blood donor
screening. In an analysis of approximately 14 million blood donations in 2008, the
overall seroprevalence was 1/27,500, with the highest rates in Florida (1/3,800), fol-
lowed by California (1/8,300) (293). Of 104 T. cruzi-infected donors with epidemiological
data, 29 (28%) were born in Mexico, 27 (26%) in the United States, 17 (16%) in El
Salvador, and 11 (11%) in Bolivia; the remaining 20 donors were born in nine other
countries of Central and South America. In a subsequent study of 22 million donations
collected between 2007 and 2011, 717 donations were confirmed seropositive by RIPA,
corresponding to a seropositivity rate of 1/31,000 (215). Among 263 donors who
provided 30-ml blood specimens, 18 (6.8%) had positive results by hemoculture and 17
had parasite genotyping results. Only 2 (1.3%) of 157 donors from areas where TcI
predominates (Mexico, Central America, and northern South America) had positive
hemocultures (both TcI). In contrast, T. cruzi grew in cultures from 13 (34.2%) of 38
donors from the Southern Cone (1 TcII, 10 TcV, 1 TcVI, and 1 not typed). Three donors
born in the United States had positive results by hemoculture, two TcV and one TcVI;
no data were available to determine the likely source of their infections. Together with
the predominance of Southern Cone donors implicated in recognized transfusion
transmissions, these data support the hypothesis that TcII/V/VI infections result in
higher parasite loads and, therefore, higher blood-borne transmission risk than TcI
infections (215, 286).
As of 26 October 2019, a total of 2,435 confirmed seropositive donors in 47 states
have been detected in screening (AABB Chagas Biovigilance Network, Chagas data
reports through 26 October 2019; www.aabb.org/programs/biovigilance/Pages/chagas
.aspx). Over the period from 2007 to 2016, the mean prevalence in first-time donors was
64 per million donors overall and 3.64 per 10,000 donors in southern California and
showed a nonsignificant decreasing trend (277). The highest number of positive
donations by calendar year was 420 in 2008; since 2014, yearly detections have ranged
from 84 to 98 (Chagas data reports mentioned above).
Organ Donor-Derived Transmission and Organ Donor Screening

A total of 14 investigations, involving organs from 14 T. cruzi-infected donors trans-
planted to 32 recipients between 2001 and 2011, were reported in a recent review (28).
Transmission occurred to 9 recipients of organs from six donors; no transmission
occurred from the remaining 8 donors (Table 10) (28). Transmission risk differs by organ
type: 3 (75%) of 4 heart, 2 (20%) of 10 liver, and 2 (13%) of 15 kidney recipients became
infected.
The earliest reported instances of transmission in 2001 and 2006 were not suspected
until at least one recipient presented with symptomatic acute Chagas disease (294,
295). More recently, some organ procurement organizations have begun selective or
universal screening of donated organs (30, 296). Four subsequent published transmis-
sion events (in a liver recipient in 2006, two heart recipients in 2006 and 2010, and a
bilateral lung recipient in 2011) were detected through systematic laboratory monitor-
ing. Three of these patients were treated and survived their T. cruzi infection (28). The
bilateral lung transplant recipient died 2 years posttransplantation from respiratory
failure; his T. cruzi PCR was intermittently positive despite prolonged benznidazole
therapy, and Chagas disease was considered a possible contributing factor in his death
(28, 297).
In the United States, screening of donors has been based largely on risk assessment;
donors born in or with significant periods of residence in Latin America, born of women
from Latin America, and/or noted to have clinical findings such as cardiomegaly
consistent with Chagas disease should be screened by IgG serology (30). Some organ
procurement organizations contract with a blood bank for donor testing, as the Abbott
PRISM and Ortho ELISA have approval for use in specimens from living and cadaveric
organ donors (231).
The recipient of an organ from an infected donor should be monitored by micros-
copy and/or PCR in serial specimens (28, 30). Seroconversion may be delayed or never

TABLE 10 Published reports of organ transplant-derived cases of Chagas disease in the United States
State of
Yr organ harvest Donor origin Implicated organ Recipient characteristics and outcome Reference
2001 GA El Salvador Kidney-pancreas 37-yr-old female with fever 6 wks posttransplant and 294
T. cruzi in blood smear, died of Chagas
myocarditis 7 mo posttransplant despite
prolonged course of nifurtimox
2001 GA El Salvador Kidney 69-yr-old female, asymptomatic, T. cruzi hemoculture 294
positive, diagnosis sought because of recipient 1
above, treated with nifurtimox, survived
2001 GA El Salvador Liver 32-yr-old female, asymptomatic, T. cruzi hemoculture 294
positive, diagnosis sought because of recipient 1
above, treated with nifurtimox but died of
unrelated causes
2005 CA U.S.-born (mother Heart 64-yr-old male with anorexia, fever, diarrhea, 295
from Mexico) diagnosed with organ rejection and treated with
steroids, T. cruzi found in blood smear 8 wks
posttransplant, PCRs became negative on
nifurtimox, died of rejection 20 wks posttransplant
2006 CA El Salvador Heart 73-yr-old male with fever, fatigue, rash, T. cruzi in 295
blood smear 7 wks posttransplant, parasitemia
cleared with nifurtimox, switched to benznidazole
because of tremors, died of heart failure 25 wks
posttransplant
2006 PA Bolivia Liver 56-yr-old male, T. cruzi detected in PCR monitoring, 28
died from gastrointestinal bleed 244 wks
posttransplant
2006 PA Bolivia Bilateral kidney 73-yr-old female, T. cruzi detected in PCR 28
monitoring, died from kidney failure 15 wks
posttransplant
2010 NY Mexico Heart 20-yr-old female, T. cruzi detected in PCR monitoring 28
and successfully treated with benznidazole,
survived at least to 24 mos posttransplant
2011 TN El Salvador Bilateral lung 36-yr-old male with cystic fibrosis, T. cruzi detected 297
in PCR monitoring, completed course of
benznidazole but had intermittent positive PCRs
posttreatment, Chagas disease possible
contributing factor to death 2 yrs posttransplant
occur in immunocompromised individuals. Positive results by PCR occur days to weeks

before parasites are detectable by microscopy (218). The incubation period of transplant-
transmitted T. cruzi infection is typically 2 to 3 months, but detection may be delayed
as long as 6 months (28). A frequently recommended monitoring schedule consists of
weekly specimens for 2 months, every 2 weeks up to 4 months, and then monthly
afterwards (Table 6) (28, 30). In the absence of other indications and assuming no
evidence of infection has been detected, the monitoring interval can be lengthened
after 6 months posttransplantation.
Chagas Cardiomyopathy and Heart Transplantation for Chagas Heart Disease in

the United States
Chagas heart disease has been recognized in U.S. health care facilities for nearly
30 years (298). Ten years ago, we estimated that there were 30,000 to 45,000 patients
with Chagas cardiomyopathy in the United States (299). Recent studies have confirmed
that Chagas disease is frequent among Latin American-born patients with cardiac signs,
including 13% (5/39) of patients with left ventricular ejection fraction (LVEF) of ⬍45%
without evidence of ischemic heart disease in New York City (300), 19% (25/135) of
patients with LVEF of ⬍40% without evidence of ischemic heart disease in Los Angeles
(301), 5.3% (17/327) of patients with any bundle branch block in Los Angeles (302), and
7.5% (6/80) of patients with pacemaker implantation in Los Angeles (303). As in studies
from Latin America (304, 305), the most common conduction system abnormalities
were right bundle branch block, left anterior hemiblock, and the combination of the

two (302, 306), and Chagas cardiomyopathy was associated with more rapid progres-
sion than other cardiac etiologies to severe disease requiring transplantation or result-
ing in death (301, 306, 307). Data from 17 Texas blood donors suggest that locally
acquired Chagas disease can also result in cardiomyopathy, but data are insufficient to
assess the relative risk for autochthonous versus imported infection (281).
In Brazil and Argentina, heart transplantation is an accepted modality to treat
end-stage Chagas cardiomyopathy, and survival for those transplanted for Chagas heart
disease is the same or better than that of recipients of cardiac transplants for other
etiologies (200, 201, 308, 309). The incidence of T. cruzi reactivation in Latin American
heart transplant cohorts varies widely, from 20 to 90% (225). In the United States, data
are published for 40 patients who underwent heart transplantation for end-stage
Chagas cardiomyopathy since 2006 (199, 225). In one review from a Los Angeles
medical center, 31 of 405 patients who received heart transplants between 2006 and
2012 were born in Latin America; 20 of the 31 had serological testing for T. cruzi and
11 (2.7% of the total number of 405) had positive results (199). Only two of the T.
cruzi-infected transplant recipients received their diagnosis prior to the transplant, both
in their home countries. Two (18.2%) patients were diagnosed with T. cruzi reactivation
when they experienced dysfunction of the transplanted heart; their infections had not
previously been suspected, and one died of cardiogenic shock. One additional patient
was asymptomatic but treated based on the finding of parasites in the explanted heart.
One of the patients in the Los Angeles cohort is included in the CDC’s comprehensive
review of 31 patients that underwent heart transplantation for Chagas cardiomyopathy
from 2012 to 2016; 19 (61%) had reactivation (225). In the CDC review, reactivation was
defined by rising parasite load by quantitative PCR in peripheral blood, a finding that
precedes both microscopically detectable parasitemia and the development of symp-
toms (218). Only one instance of reactivation was symptomatic at the time of diagnosis,
and all patients with reactivation were alive at the end of follow-up.
Congenital Chagas Disease in the United States

Based on births to Latin American-born women in the United States and T. cruzi
infection prevalence in their home countries, it was estimated that between 60 and 315
congenitally infected infants are born in the United States each year (299). Neverthe-
less, only two congenital infections have been reported, both in infants of Bolivian
women (310, 311). The first infant was delivered by caesarian section at 29 weeks
gestation because of fetal hydrops (a classic presentation of severe congenital Chagas
disease) (18). The diagnosis was not suspected until the mother reported a prior
diagnosis of Chagas disease (311). The second infant was also delivered by caesarian
section at 30 weeks due to placental abruption and had a pericardial effusion, ascites,
and respiratory distress requiring intubation until the 10th day of life (310). The
diagnosis was made on histopathological examination of the placenta. Both infants
were successfully treated with antitrypanosomal therapy.
SPECIAL CONSIDERATIONS IN THE UNITED STATES

An adequate public health response to Chagas disease in the United States faces
critical challenges, including limited patient and provider awareness of the disease,
societal, economic, and health system barriers to patient access, and sparse diagnostic
options with an inadequate evidence base to assess performance.
Physician Awareness
Surveys indicate that the majority of physicians practicing in the United States have
limited knowledge of Chagas disease and seldom consider the diagnosis, even when
caring for Latin American-born patients at high risk or with typical clinical syndromes
(312, 313). In one survey, 23% of cardiologists, 47% of obstetrician/gynecologists, and
25% of transplantation specialists reported that they had never heard of Chagas disease
(312). In a survey of more than 400 obstetrician/gynecologists, 78% reported that they

never considered the diagnosis of Chagas disease in their Latin American patients and
fewer than 10% were cognizant of the risk of vertical transmission to the infant (313).
Patient Awareness and Access

Awareness and knowledge of Chagas disease are also limited among those at risk.
Of Latin American immigrants interviewed in community outreach settings in southern
California, 86% had never heard of Chagas disease, even though 62% reported having
seen triatomines in their countries of origin (314). Patients with Chagas disease en-
counter multiple barriers to health care access in general, not just for Chagas disease:
these patients disproportionately live below the poverty line, have less than a high
school education, lack health insurance, and have difficulty taking time off from work
for medical appointments (315). Even legal immigrants have lower access than citizens
to publicly funded health insurance in most states; concerns about revealing immigra-
tion status may make patients reluctant to seek care (315).
Diagnostic Issues
There are currently four commercial IgG serological tests cleared by the FDA for
diagnostic use in the United States, including three ELISA kits (Hemagen [Hemagen
Diagnostics, Waltham MA], Chagatest Recombinante 3.0 [Wiener Laboratories, Rosario,
Argentina], and Ortho [Ortho-Clinical Diagnostics, Inc., Raritan, NJ]) and one point-of-
care test (ChagasDetectPlus [InBios International, Seattle, WA]) (316). With the excep-
tion of the Ortho ELISA, which is also licensed for blood donor screening (215, 277, 317),
there is a paucity of data on the performance of these assays in specimens from
populations in the United States or in the likely predominant countries of origin of
infected U.S. residents (Mexico, El Salvador, Guatemala, and Honduras) (273, 293, 299).
Discordant serology has been reported as a particular problem in Mexico (318). Some
recombinant tests with excellent performance in the Southern Cone show discordance
or low sensitivity when applied in some areas where TcI is predominant (319, 320). In
addition, no commercial laboratory in the United States currently offers more than one
validated IgG serological assay. The diagnosis of chronic T. cruzi infection requires
positive results by two distinct IgG assays and is therefore not possible with commercial
testing alone (16). Currently, the only laboratory that conducts multiple IgG serological
assays under Clinical Laboratory Improvement Amendments (CLIA) is the CDC Division
of Parasitic Diseases and Malaria (DPDM) laboratory.
FDA Approval of Benznidazole and Resulting Changes

Until May 2018, when benznidazole became commercially available, prescribing antit-
rypanosomal treatment necessitated a consultation with CDC epidemiologists (321).
Confirmation of the diagnosis, recommendations for treatment, and advice on moni-
toring and management of side effects were necessary components of these consul-
tations, and reporting of adverse events was mandatory under the investigational
protocol. Ensuring appropriate diagnostic confirmation, judicious treatment decisions,
and adequate side effect monitoring going forward will require efforts to raise provider
awareness and knowledge about Chagas disease and antitrypanosomal therapy.
From October 2011 to May 2018, the CDC released benznidazole for 365 patients
with confirmed T. cruzi infection (321). Four (1.1%) patients had acute-phase infection,
two organ derived, one congenital, and one presumed to be vector borne. Treatment
was administered for T. cruzi reactivation in 35 (9.6%) patients, comprising 29 organ
transplant recipients, 5 HIV-coinfected patients, and 1 on chemotherapy for malig-
nancy. The vast majority of patients were adults, 236 (64.7%) aged 19 to 50 and 97
(26.6%) older than 50 years. Only 2 (0.5%) of 365 treated patients were aged 2 to
12 years, the age range for which the FDA approved benznidazole.
Insud Pharma’s approach to FDA approval and drug marketing represents a new
paradigm in the United States, with patient access and affordability as central concerns
(250). The FDA Priority Review Voucher (PRV) program was established in 2008 to
provide an incentive for new drug development for neglected tropical diseases (NTD)

but has had limited impact and unintended negative consequences (322). In one recent
example, FDA approval of miltefosine, a drug used for leishmaniasis, was followed by
an astronomical price increase, and the company ceased production after receiving and
selling the PRV, leading to a global shortage (323). In contrast, the Insud Pharma/Exeltis
Patient Assistance Program, funded in part by the benznidazole PRV, ensures that the
cost to the patient will not exceed $60 per course (250).
Public Health Surveillance and Response

As of 2017, Chagas disease was a reportable condition in six states, Arizona,
Arkansas, Louisiana, Mississippi, Tennessee, and Texas (324); Utah added Chagas dis-
ease to its notifiable disease list recently. All of these states except Arkansas and Utah
have had published reports of locally acquired T. cruzi infection (50–52, 280, 282, 283).
Most of the states focus on autochthonous transmission and incident cases, and several
have provisions for submission of triatomines for identification (324). In Texas, the
Department of State Health Services (DSHS) provides extensive on-line guidance and
data to health care providers and the public, including a summary of reported Chagas
disease case data; of 124 cases reported from 2013 to 2017, all were chronic infections
and 22 were judged to have resulted from local transmission (325). These figures likely
overlap substantially with the published locally acquired infections detected in blood
donors in recent years (282, 283). The Texas Chagas Taskforce, which includes the DSHS
and several universities, addresses many public health aspects of Chagas disease,
including provider and patient resources and educational materials on local vectors
(326).
PUBLIC HEALTH APPROACHES

Public health approaches to Chagas disease comprise primary prevention (preven-
tion of transmission), secondary prevention (early treatment of infection to prevent
sequelae), and tertiary prevention (medical and surgical management of morbidity to
improve survival and quality of life) (Table 11) (309). In Latin America, primary preven-
tion through vector control is responsible for the vast majority of the decrease in
estimated annual incidence from 500,000 in 1991 to 30,000 today (35). Primary pre-
vention through vector control is virtually impossible in the United States, since the
vectors are sylvatic; however, barriers to house invasion, elimination of microhabitats
close to houses, and improved awareness among those living in areas of risk could help
decrease the likelihood of autochthonous transmission. Blood bank screening has also
been highly successful in both Latin America and the United States. The last detected
blood component-derived infection in the United States occurred in 2006, before
screening was instituted (277, 287).
In Latin America, secondary prevention efforts include congenital Chagas disease
screening programs and mass testing and treatment of children in high-prevalence
zones (263, 327). Prenatal and congenital screening programs are attractive for several
reasons. Cure rates are ⬎90% in infected infants and drug tolerance is excellent (257,
258); treatment of infected women, once lactation ends, significantly decreases the risk
of transmission in future pregnancies (191); and detection of maternal infection pro-
vides a screening opportunity for her other children, who are also at risk for T. cruzi
infection (192). However, congenital screening programs are also complicated. With
current diagnostic modalities, effective congenital Chagas disease screening requires a
multistep algorithm consisting of prenatal serology in women and testing of infants
of seropositive mothers 2 or 3 times (parasitological or molecular testing in the first
months of life, and for those who test negative, serological testing at 9 to 12 months)
(216). Even in Bolivia, where infection prevalence in women is often 15% or higher and
awareness is high, congenital Chagas disease detection is challenging, because of low
sensitivity of microscopy and ⬎80% loss to follow-up for the 9- to 12-month visits (328).
A pilot study in a hospital in Houston screened 4,000 women, 75% of whom were
born in Latin America (329). Ten (0.25%) women had confirmed T. cruzi infection; no
infected infants were detected. A recent analysis concluded that in the United States,

TABLE 11 Public health screening options for Chagas disease in the United States
Bern et al.
Published estimates of
Intervention details and screening yield
Target population Screening method(s) Primary goal Secondary goal effectiveness (reference[s])
Blood donors Serologic screening Prevent transmission Refer infected persons for Discard screen-positive donations; ⬃1/15,000 first-time donors,
management highly effective up to 1/2,700 in high-risk
area (277)
Organ donors Serologic or risk-based Prevent transmission Heart from infected donor not used, 0.9% in combined risk-based
screening other organs used with and serologic donor
appropriate monitoring; highly screening (296)
effective
Pregnant women from Maternal serology, Detect infected infants Refer infected women Early treatment of infants, treatment
January 2020 Volume 33 Issue 1 e00023-19

⬃10 mothers, ⬍1 infected
Latin America; infants serial testing of early in life and their other of women after lactation ends, child per 4,000 high-risk
of infected women infants, serology in children for treatment treatment of infected siblings; women (majority born in
siblings highly effective in infants and Latin America) (329)
children, moderately effective in
young women
Latin American Serologic screening Detect asymptomatic Treatment of infected individuals; 0.5–1% in high-risk
immigrants and confirmatory infected individuals effectiveness high in children, populations; many of
testing uncertain in adults those detected were ⬎50
yrs old, for whom
treatment is not generally
recommended (273, 274)
Patients from Latin Serologic screening Detect symptomatic Standard cardiac management, Latin American-born patients
America with and confirmatory infected individuals prospective monitoring for with bundle branch blocks,
nonischemic cardiac testing reactivation in transplant 5%; pacemakers, 7.5%;
syndromes recipients; effective in improving depressed left ventricular
survival and quality of life ejection fraction, 13–19%
(300–303)
cmr.asm.org 30
Clinical Microbiology Reviews
universal congenital Chagas disease screening and treatment would be cost saving
with congenital transmission rates of ⱖ0.001% and maternal prevalence of ⬎0.06%
(330). The results vary substantially depending on the cost and performance of the
maternal screening test; thus, it will be essential to ascertain the currently unknown
sensitivity of available serological assays in at-risk populations in the United States.
The effectiveness of secondary prevention strategies depends strongly on the
effectiveness of antitrypanosomal treatment in preventing the development and pro-
gression of cardiac disease, which remains a controversial topic without a clear answer
(269). Current practice in Latin America prioritizes the diagnosis and treatment of
children 15 years old or younger, but the vast majority of infected individuals in the
United States are adults. In the sparse available community screening data, the T. cruzi
prevalence in Latin American adults 40 years old or younger was 0.64 to 0.95%, compared
to 1.42 to 1.78% among those older than 40 years (273, 274). In limited community
screening to date, no infections have been detected among children (274).
Tertiary prevention has had a major impact on the survival and quality of life of
persons living with T. cruzi infection in Latin America (331), and the experience of a
dedicated center of excellence based in the cardiology service of a large Los Angeles
hospital confirms this model in the United States (301, 302, 332–334). Expanding this
effort will require outreach efforts to cardiologists and primary care physicians and
making accurate diagnostic testing more widely available.
CONCLUSIONS
Chagas disease causes disease of the heart and/or gastrointestinal tract in 20 to 30%
of those infected by T. cruzi. The southern half of the United States contains enzootic
cycles of T. cruzi, involving 4 major and 7 minor triatomine vector species. T. cruzi
infection has been detected in multiple mammalian species, including raccoons, opos-
sums, woodrats, and dogs. Locally acquired Chagas disease has been increasingly
recognized in the United States over the past 10 years, largely due to screening of
blood donations and investigations of infected blood donors without exposure in Latin
America. Nevertheless, imported chronic T. cruzi infections in migrants from Latin
America vastly outnumber autochthonous human cases, and locally acquired infection
is rarely detected in the acute phase. Benznidazole is now FDA approved as a treat-
ment, and clinical and public health efforts are under way by researchers and some
state health departments to broaden access to diagnosis and treatment.
Making progress will require work on many fronts, including innovative ways to
improve the knowledge base among providers, expand the availability of high-quality
diagnostic and confirmatory testing, and pilot public health screening data to develop
evidence-based targeting strategies. However, increased awareness of Chagas disease
is crucial to all aspects of this effort. Providers with awareness of the disease can screen
those at risk when they present for clinical care, with the highest priority for children
and women of child-bearing age, since the benefit of antitrypanosomal therapy is clear
for these groups. The appropriate index of suspicion saves lives when reactivation of
chronic infection and donor-derived T. cruzi infection are recognized in a timely fashion.
Diagnosis of T. cruzi infection and follow-up for the onset or progression of cardiomy-
opathy or gastrointestinal disease can mitigate morbidity and improve survival and
quality of life.
ACKNOWLEDGMENTS
We thank Ernesto Barrera Vargas, Rochelle Hoey-Chamberlain, Christiane Weirauch,
Gena Lawrence, and Sonia Kjos for images of the triatomine vectors and Henry Bishop
for images of T. cruzi.
Caryn Bern received consulting fees from Exeltis in 2017 and 2018. James H. Maguire
received consulting fees from Bayer in 2017. Louisa A. Messenger and Jeffrey D.
Whitman report no financial interests.

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Caryn Bern is Professor of Epidemiology Jeffrey D. Whitman is currently a postdoc-

and Biostatistics in the School of Medicine, toral research fellow in the Department of
University of California, San Francisco. She Laboratory Medicine at the University of Cal-
holds an M.D. from Stanford University School ifornia, San Francisco, School of Medicine. He
of Medicine and an M.P.H. from the Johns has an M.S. in biochemistry and molecular
Hopkins University School of Public Health biology from the University of California, Riv-
and is board certified in internal medicine. erside, and an M.D. from the David Geffen
She was a medical epidemiologist at the School of Medicine at UCLA. His research in-
Centers for Disease Control and Prevention terests include improving diagnostic dispari-
from 1990 to 2011 and worked for 15 years ties in global health settings with a method-
in the CDC Division of Parasitic Diseases and ological focus on mass spectrometry-based
Malaria. Dr. Bern has worked on clinical and epidemiological aspects of metabolomics and proteomics. He is currently pursuing research related to
Chagas disease since 2003, with projects in Peru, Bolivia, and the United improving Chagas disease diagnostics.
States. Dr. Bern’s current research focuses on diagnosis, treatment,
epidemiology, and control of Chagas disease and visceral leishmaniasis.
James H. Maguire is Professor of Medicine,
Harvard Medical School, and Senior Physi-
Louisa A. Messenger is an Assistant Profes- cian, Brigham and Women’s Hospital, Boston,
sor in the Department of Disease Control of MA. He completed his M.D. from Harvard Med-
the London School of Hygiene and Tropical ical School and M.P.H. from Harvard School of
Medicine. She holds a B.A. from the Univer- Public Health and is board certified in internal
sity of Cambridge and an M.Sc. in Medical medicine and infectious diseases. He was the
Parasitology and Ph.D. in Molecular Biology chief of CDC Parasitic Diseases Branch from
from the London School of Hygiene and 2001 to 2005. He was Professor and Head,
Tropical Medicine. She has been a recipient of Division of International Health, Department of
the L’Oréal-UNESCO For Women in Science UK Epidemiology and Preventive Medicine, Uni-
and Ireland Fellowship and an American Soci- versity of Maryland School of Medicine from 2005 to 2008. Dr. Maguire
ety for Microbiology/Centers for Disease Con- performed seminal research in the epidemiology and cardiac manifesta-
trol and Prevention Fellowship. Dr. Messenger has lived and worked tions of Chagas disease in rural Brazil in the 1980s and collaborated on
extensively in South America and Africa to improve methods of Chagas studies of Chagas disease in Peru and Bolivia from 2003 to the present.
disease and malaria control, respectively.

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REVIEW

Chagas Disease in the United States: a Public Health

January 2020 Volume 33 Issue 1 e00023-19 Clinical Microbiology Reviews cmr.asm.org 1

SPECIAL CONSIDERATIONS IN THE UNITED STATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

SUMMARY Trypanosoma cruzi is the etiological agent of Chagas disease, usually

BIOLOGY AND TRANSMISSION OF TRYPANOSOMA CRUZI

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 2

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 3

EPIDEMIOLOGY AND ECOLOGY

Triatomine Vector Biology

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 4

TABLE 1 Countries in which Chagas disease is endemic, estimates of national

Central America Belize 0.330 1,040

South America Argentina 3.641 1,505,235

Total 1.056 5,742,167d

Triatomine Distribution in the United States

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 5

TABLE 2 Triatomine vectors in the United Statesa

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 6

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 7

Allergic Reactions to Triatomine Antigens

Wild and Domestic Animal Reservoirs

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 8

epidemiological importance of particular species is highly variable, depending on local

Transmission Potential in the United States

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 9

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 10

Each T. cruzi DTU is characterized by distinct but often overlapping transmission

Trypanosoma cruzi Molecular Epidemiology in the United States

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 11

eAL, FL, GA, KY, LA, NC, TN, VA.

fIN, KS, MO, OH, OK.

Issues Underlying T. cruzi Genotyping Data Collection

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 12

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 13

infected patients, direct genotyping is insensitive but may be improved if multiple

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 14

the most common gene sequence is ampliﬁed in a reaction. However, recently, a

Chronic T. cruzi Infection

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 15

TABLE 6 Chagas disease diagnostic testing by clinical context

Persons at risk for acute T. cruzi infection

Persons at risk for T. cruzi reactivation

cPCR is substantially more sensitive than microscopy in peripheral blood.

Gastrointestinal involvement is much less frequent than cardiomyopathy. Esopha-

Chagas Disease in the Immunocompromised Host

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 16

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 17

immunosorbent assays (ELISAs), immunoﬂuorescence assays (IFAs), and immunochro-

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 18

ETIOLOGICAL TREATMENT AND CLINICAL MANAGEMENT

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 19

Acute and Congenital T. cruzi Infection

Treatment of Chronic T. cruzi Infection

January 2020 Volume 33 Issue 1 e00023-19 cmr.asm.org 20

May be offered with consideration of potential risks and beneﬁts,

Recommendation against treatment

Management of the Immunocompromised Host

HUMAN CHAGAS DISEASE IN THE UNITED STATES