Small Animals

JAVMA—18-09-0496—Madigan—1 fig—4 tab—LJP—RES

Investigation of a combination of amiodarone
and itraconazole for treatment of American trypanosomiasis
(Chagas disease) in dogs

Roy Madigan dvm OBJECTIVE

To evaluate clinical, serologic, parasitological, and histologic outcomes of
Sean Majoy dvm, ms dogs with naturally occurring Trypanosoma cruzi infection treated for 12
Kristine Ritter bs months with amiodarone and itraconazole.
Juan Luis Concepción phd ANIMALS
María Elizabeth Márquez msc 121 dogs from southern Texas and southern Louisiana.
Sasha Caribay Silva bsc PROCEDURES
Chih-Ling Zao phd Treatment group dogs (n = 105) received a combination of amiodarone hy-
drochloride (approx 7.5 mg/kg [3.4 mg/lb], PO, q 24 h, with or without or
Alexandra Pérez Alvarez bsc a loading dosage protocol) and itraconazole (approx 10 mg/kg [4.5 mg/lb],
Alfonso J. Rodriguez-Morales md, hondsc PO, q 24 h, adjusted to maintain a plasma concentration of 1 to 2 µg/mL)
for 12 months. Control group dogs (n = 16) received no antitrypanosomal
Adriana C. Mogollón-Mendoza md medications. Serologic assays for anti–T cruzi antibodies, PCR assays for T
J. Scot Estep dvm cruzi DNA in blood, and physical evaluations were performed 1, 6, 9, 12, and
Gustavo Benaím phd 24 months after study initiation (baseline). Adverse events were recorded.
Outcomes of interest were recorded and compared between groups.
Alberto E. Paniz-Mondolfi md, phd
RESULTS
From Animal Hospital of Smithson Valley, 286 Singing
Oaks, Ste 113, Spring Branch, TX 78070 (Madigan, Estep); 86 of 105 treatment group dogs and 8 of 16 control group dogs survived and
LTC Daniel E. Holland Military Working Dog Hospital, completed the study (5/19 and 6/7 deaths of treatment and control group
1219 Knight St, Joint Base San Antonio-Lackland, Lack- dogs, respectively, were attributed to T cruzi infection). Mean survival time
land AFB, TX 78236 (Majoy, Ritter); Laboratorio de Enzi- until death attributed to T cruzi was longer (23.19 vs 15.64 months) for
mología de Parásitos, Facultad de Ciencias, Universidad
de Los Andes, Mérida 5101, Mérida, Venezuela (Concep- the treatment group. Results of PCR assays were negative for all (n = 92)
ción, Márquez, Silva, Paniz-Mondolfi); VRL Laboratories, tested treatment group dogs (except for 1 dog at 1 time point) from 6
7540 Louis Pasteur, Ste 250, San Antonio, TX 78229 to 24 months after baseline. Clinical improvement in ≥ 1 clinical sign was
(Zao); Infectious Diseases Research Branch, Venezuelan observed in 53 of 54 and 0 of 10 treatment and control group dogs, respec-
Science Incubator and the Zoonosis and Emerging Patho-
gens Regional Collaborative Network, Av Intercomunal tively; adverse drug events were minor and reversible.
Barquisimeto-Acarigua, Urb Los Rastrojos, Cabudare
3023, Lara, Venezuela (Pérez Alvarez, Mogollón-Mendoza, CONCLUSIONS AND CLINICAL RELEVANCE
Paniz-Mondolfi); Grupo de Investigación Salud Pública e Results suggested efficacy of this trypanocidal drug combination for the treat-
Infección, Facultad de Ciencias de la Salud, Universidad ment of T cruzi infection in dogs. (J Am Vet Med Assoc 2019;255:xxx–xxx)
Tecnológica de Pereira, Sector La Julita, Pereira 660003,
Risaralda, Colombia (Rodriguez-Morales); Decanato
de Ciencias de la Salud, Escuela de Medicina, Uni-
versidad Centroccidental Lisandro Alvarado, Barqui-
simeto 3001, Lara, Venezuela (Mogollón-Mendoza); tors known as kissing bugs, which are prevalent in
Laboratorio de Señalización Celular y Bioquímica de North and South America.3 Defecation by infected
Parásitos, Instituto de Estudios Avanzados, Carretera
Nacional Hoyo de la Puerta, Sartenejas, Caracas 1015- insects during or after a blood meal passes infective
A, Venezuela (Benaím, Paniz-Mondolfi); Instituto de trypomastigotes into the bite wound.3 Other routes of
Biología Experimental, Facultad de Ciencias, Univer- infection include transplacental transmission to off-
sidad Central de Venezuela, Colinas de Bello Monte,
Caracas 1041-A, Venezuela (Benaím); IDB Biomedical spring of infected pregnant women and bitches, oral
Research Institute, Av Intercomunal Barquisimeto- ingestion (including consumption of infected meat),
Acarigua, Urb Los Rastrojos Cabudare 3023, Lara, breastfeeding, and blood transfusion.3–5 After trypo-
Venezuela (Paniz-Mondolfi); and Instituto Venezolano
de los Seguros Sociales, Direccion de Salud/Direccion mastigotes enter the circulation, there is a brief pe-
Nacional de Docencia e Investigación, Edif Sede, Cara- riod of replication and hematogenous spread through
cas 1010, Venezuela (Paniz-Mondolfi).
mononuclear cells, with the final destination being
Address correspondence to Dr. Madigan (roytmadigan@ various tissues in the host, including the heart, fat,
yahoo.com).
and the lymphatic system, where they reside and mul-
tiply as intracellular amastigotes.3
A merican trypanosomiasis, commonly known as
Chagas disease, is a parasitic condition caused
by the hemoflagellated protozoa, Trypanosoma cru-
A century after its discovery, T cruzi infection
remains a serious health problem. It is a leading cause
of cardiomyopathy that affects > 8 million people
zi.1,2 Active transmission is through triatomine vec- worldwide.1 It is predicted that Chagas disease will
ABBREVIATIONS cause at least 200,000 human deaths globally over
DTU Discrete typing unit the next 5 years, which is identical to the number of
IFA Immunofluorescence antibody test women living in the United States predicted to die of
MANCOVA Multivariate ANCOVA breast cancer in the same time period.2,6

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Whereas the highest prevalence of cardiomyopa- infection are the same as those found for a number
thy due to Chagas disease in people is found in Cen- of other common heart abnormalities, including di-
tral and South American countries, studies7–9 show a lated cardiomyopathy and chronic valvular disease.20
widespread distribution of American trypanosomia- Therefore, cardiomyopathy attributable to American
sis in dogs in Texas, Louisiana, and Georgia and in- trypanosomiasis may be difficult to differentiate from
dicate that these southern states are the sites of im- that resulting from other causes, especially in breeds
portant reservoirs for transmission of Trypanosoma that are predisposed to these types of acquired heart
cruzi in the United States. The prevalence of T cruzi dysfunction.20
infection in stray and shelter-housed dogs has been Information on survival time in chronically in-
reported to be as high as 7.5% to 8.8% and may ex- fected untreated dogs is sparse. However, there is
ceed 50% in some working dog kennels in Texas.10,11 likely to be a distinct difference in prognosis accord-
On the basis of a 2012 AVMA estimate12 of the canine ing to age, in view of 1 study21 in which investigators
population in Texas, these findings suggest there found dogs identified as having T cruzi infection at
could be approximately 630,000 dogs with T cruzi a mean age of 9 years survived for 30 to 60 months,
infection in that state alone.9,10 A similar prevalence whereas those that had the diagnosis made at a young-
of T cruzi infection (ie, > 50% in hunting dogs in the er age (mean, 4.5 years) survived for a maximum of 5
region and 22% in its general canine population) has months after diagnosis.
been reported in southern Louisiana.7 There are presently no drugs approved by the US
The pattern of disease progression in dogs with FDA for the treatment of T cruzi infection in dogs.
American trypanosomiasis is similar to that in peo- Currently available specific anti–T cruzi chemothera-
ple, which comprises acute, chronic asymptomatic, peutic agents have unsatisfactory results in people
and chronic symptomatic phases.13–15 Acute disease and in dogs.22,23 Much consideration has been given
occurs within 3 weeks after inoculation and centers to nitroimidazole- and nitrofuran-derived drugs, such
around myocarditis with severe inflammation follow- benznidazole and nifurtimox, which were developed
ing the death of cardiac cells.16,17 Most affected dogs in the 1960s and 1970s24–27 and are not presently com-
are < 1 year of age, and clinical signs commonly in- mercially available in the United States. These com-
clude tachyarrhythmias, respiratory distress, lymph- pounds act by generation of free radicals and damage
adenopathy, anorexia, diarrhea, neurologic abnor- the DNA of the parasite28 but have several major limi-
malities, peripheral edema, collapse, and sudden tations including lack of efficacy in 80% to 100% of
death.13–15 Parasitemia is profound and the mortality patients in the chronic phase of the disease, inability
rate is high, especially among < 1-year-old dogs.13,14 to prevent progression of chronic cardiomyopathy in
A chronic phase without overt clinical signs, human patients with the disease,23,27,28 and an intrin-
often described in the literature as the chronic as- sic resistance to benznidazole in some major strains
ymptomatic phase, is most commonly encountered of T cruzi.29,30
in dogs.14,18 This phase is characterized by a slowly Many compounds with proven trypanocidal
progressing, fibrosing inflammatory process in the activity are currently under development or being
heart and variable shedding of the organism into the tested.24,31,32 Among these are new nitroimidazole de-
circulation.14,18 Although sudden death is a possibility rivatives such as fexinidazole; ergosterol biosynthesis
at this stage, particularly as a result of fatal exercise- inhibitors; and experimental drugs such as cysteine
induced or excitement-induced arrhythmias, most proteases and inhibitors of trypanothione metabo-
signs such as mild lethargy and cardiac conduction lism, kinetoplastid proteasomes, polyphosphate me-
disturbances are clinically subtle and can be over- tabolism, and purine synthesis as well as oxaborole,
looked by the owner.14,18,19 fenarimol, and amidine analogs and derivatives.32
The proportion of cases of T cruzi infection that However, very few of these drugs, such as allopurinol
progress to a chronic phase with overt clinical signs and the azole derivatives itraconazole, fluconazole,
in dogs is unknown; however, approximately 30% of ketoconazole, posaconazole, and ravuconazole, have
cases progress to the chronic symptomatic stage in been tested in human clinical trials.32
human patients.3,17 Electrocardiographic abnormali- Antifungal azoles work by disrupting the path-
ties were found in 5 of 16 (31%) infected dogs in ex- ways for biosynthesis of sterols,33 and because
perimental studies14,19; these findings were mostly at- T cruzi and fungi share similar pathways for biosyn-
tributed to inflammation and fibrosis in the electrical thesis of sterols, antifungal azoles have been used as
pathways but also to selective destruction of neurons an alternative treatment for Chagas disease in human
causing parasympathetic denervation in the heart. patients.34–36 A cure rate of 33% was observed for
Also common in dogs during this phase are bilateral itraconazole-treated human patients during long-term
cardiac dilatation with associated pulmonary edema, follow-up,32,37 and lower cure rates of 8% to 31% have
pericardial effusion, and ascites; secondary mitral or been reported in patient groups treated with newer
tricuspid valve insufficiency; hepatosplenomegaly; azoles, such as posaconazole and ravuconazole.35,38
and exercise intolerance and lethargy.14,19 It is impor- A human patient in Venezuela was cured of Cha-
tant to note that the clinical signs of cardiomyopathy gas disease after receiving a combination of itracon-
in dogs that have chronic, clinically evident T cruzi azole and amiodarone.36 These 2 drugs were used to

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target specific metabolic pathways of the parasite signed to the treatment and control groups by simple
related to synthesis of ergosterol and intracellular cal- randomization.a Group allocation allowed for statis-
cium modulation.39,40 Amiodarone is an antiarrhyth- tical comparison between groups while minimizing
mic agent and the drug of choice for prevention of the number of dogs that did not receive treatment
complex arrhythmias in people with cardiomyopathy during the study.
caused by T cruzi infection.36,40,41 This agent selec-
Treatment group—All treated dogs received
tively disrupts calcium homeostasis in T cruzi,39,42 amiodarone hydrochlorideb,c by 1 of 2 protocols. The
blocks the critical protease cruzipain,43 and inhibits treatment was supplied as scored 200-mg tablets to
production of sterols.39 We became interested in the approximate the calculated dose to the nearest quar-
potential synergy that could be achieved by combin- ter tablet. One protocol included a loading dosage of
ing amiodarone with itraconazole to capitalize on 15 mg/kg [6.8 mg/lb], PO, every 12 hours for 7 days,
the ability of these drugs to inhibit sterol synthesis followed by 15 mg/kg, PO, every 24 hours for 14 days,
at different steps in the ergosterol pathway and to and then a maintenance dosage of 7.5 mg/kg [3.4 mg/
disrupt calcium homeostasis in the parasite. The pur- lb], PO, every 24 hours for the remainder of the treat-
pose of the study reported here was to evaluate the ment period (total, 12 months). The other protocol
clinical, serologic, parasitological, and histopatholog- was a maintenance dosage of 7.5 mg/kg, PO, every 24
ic outcomes of dogs naturally infected with T cruzi hours throughout the treatment period, which was
and treated with a combination of itraconazole and given to the military working dogs because of attend-
amiodarone for 12 months. We hypothesized that the ing clinician concerns about potential proarrhythmic
combination of amiodarone and itraconazole would effects of a higher initial dosing regimen. The 7.5-mg/
eliminate T cruzi parasitemia and improve clinical kg dose of amiodarone was halved if adverse events
outcomes in these dogs. occurred and were thought to be related to the drug.
Itraconazoled,e was started on the same day as the
Materials and Methods first amiodarone treatment at a dosage of 10 mg/kg (4.5
mg/lb), PO, every 24 hours. The drug was provided
Animals and study design as 100-mg capsules and rounded down to the nearest
A convenience sample of client- or government-
whole capsule, or in cases where a revised protocol
owned dogs from southern Texas and southern Loui-
was required because of low body weight, itracon-
siana that were naturally infected with T cruzi were
azole was given at approximately 10 mg/kg, PO, ev-
enrolled in the multicenter, prospective, controlled
ery 48 hours. The dosage for each dog was adjusted to
study between October 12, 2009, and December 7,
maintain a plasma itraconazole concentration of 1 to 2
2017. The client-owned dogs were patients at the first µg/mL following sample collection and testing 30 to 45
author’s hospital and any of 11 referring hospitals. days after initiation of treatment when steady state had
Owners were informed of their dogs’ positive anti– been achieved. Plasma itraconazole concentrations
T cruzi antibody status shortly after diagnosis, and were reassessed as needed for patients considered to
the dogs were randomly assigned to treatment or have suboptimal circulating drug concentrations.
control groups after owner consent was provided for A complete physical examination and blood sam-
study participation. Government-owned dogs were ple collection for a CBC, serum biochemical analysis,
from 1 military base in Texas, and inclusion of these PCR assay for T cruzi DNA, and serologic (IFA) testing
dogs in the study was approved by the director of the for antibodies against the organism were performed
Military Working Dog program. The study protocol at the start of the study, immediately prior to initia-
was approved by the Department of Defense Military tion of the drug treatments (ie, baseline). An ECG
Working Dog Facility animal use committee at Joint was obtained and echocardiography was performed
Base San Antonio-Lackland for use with government- prior to treatment initiation and during follow-up
owned dogs, and the same protocol was distributed when deemed appropriate by the attending clinician
and followed for privately owned dogs. Veterinarians and authorized by the clients (for privately owned
at each participating facility were given the study dogs). Radiographs were evaluated for patients that
protocol to execute. All data from referring practices showed clinical signs of heart disease (if authorized
and institutions were received and collated via elec- by the client for privately owned dogs) prior to and
tronic records transfer and telephone communica- on completion of the study. Follow-up visits dur-
tions. The inclusion criteria were ≥ 1 positive IFA test ing the treatment period took place 1, 6, 9, and 12
result and 1 positive result for antibody titers (≥ 1:20) months after the start of drug administration. These
by ELISA for T cruzi. Pregnant bitches were excluded dogs underwent complete physical examination and
because of concerns that treatment could potentially blood sample collection (5 mL) at all follow-up vis-
be teratogenic. Dogs that received compounded itra- its; a CBC, serum biochemical analysis, and measure-
conazole during the study were excluded from all ment of plasma itraconazole concentrations were
analyses because of bioavailability concerns.44 Demo- performed at the 1-month follow-up visit, and PCR
graphic data (age, sex, breed, and primary housing assay and serologic testing (by IFA) were performed
type [indoor vs outdoor]) as well as a detailed medi- at subsequent visits. At each visit, the owners or han-
cal history were recorded for all dogs. Dogs were as- dlers were asked whether the dogs had experienced

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any clinical signs possibly associated with T cruzi a dog that failed to respond to the study treatment, or
infection, such as weakness, vomiting, diarrhea, ab- euthanasia because of quality of life concerns.
dominal distention, collapse, or prolonged periods of
hyporexia or anorexia.9,21 Owners or handlers were Hematologic tests for T cruzi
instructed to monitor their dog’s appetite and be- PCR assays—Blood samples (5 µL) were used for
havior and to report any episodes of excessive pant- PCR assays targeting T cruzi kinetoplast minicircle DNA
ing or weight loss in addition to the aforementioned and T cruzi satellite DNA. The PCR assay methods (Sup-
signs. When adverse events plausibly attributable to plementary Appendix S1, available at avmajournals.
treatment were identified, drug doses were modified avma.org/doi/suppl/10.2460/javma.255.3.xxx) were per-
(by adjustment of itraconazole dosage on the basis of formed as previously described36,45 by study authors (CLZ
plasma drug concentrations greater than the targeted and AEP-M) at VRL Laboratories, San Antonio, Tex, or Lab-
therapeutic range or, if itraconazole concentrations oratorio de Enzimología de Parasitos, Universidad de Los
were deemed appropriate, adjustment of the amioda- Andes, Mérida, Venezuela. Blood samples were collected
rone dosage). into EDTA-containing tubes and shipped and processed
immediately or kept at 4°C for ≤ 1 week. For samples
Control group—Control group dogs underwent stored for durations > 1 week, guanidine buffer (guani-
physical assessments in the same manner as treat- dine hydrochloride; 6M; pH, 8.0; with 0.2mM EDTA) was
ment group dogs at the start of the study (baseline) added to blood samples (1:1 [vol:vol]), and the sample was
and during treatment phase follow-up visits 1, 6, 9, stored in liquid nitrogen or at −80°C until analysis. Ge-
and 12 months later. Follow-up information was ob- nomic DNA was extracted with a commercial kit,f and the
tained from owners and handlers, and monitoring PCR assaysg were performed with commercially prepared
instructions were given as for the treatment group. A primersh (for sequences proposed by Schijman et al46),
CBC and serum biochemical analysis were performed buffer solution,i and a Taq polymerase product designed
at the start of the study and at the 1-month follow-up for enhanced amplification.j Detection of both targets was
visit. Molecular assessment for parasitemia (by PCR considered a positive result.
assay) and serologic evaluation (by IFA) were per-
formed as described for the treatment group at the Serologic tests—Serum samples were obtained
time of diagnosis; PCR assays for the control group from whole blood with hospital-specific protocols at
were also performed at 6, 9, and 12 months. multiple facilities and transported (on ice in refrigerated
conditions of 1°C) to a universityk or Department of De-
Posttreatment follow-up and study end points— fensel diagnostic laboratory to be analyzed for antibodies
All surviving dogs in both groups underwent com- against T cruzi by IFA. Multiple dilutions were performed
plete physical examination and blood sample collec- on all samples, and a positive result was defined as a titer ≥
tion (5 mL) for PCR assay and IFA 12 months after 1:20. Substrate slides of whole T cruzim and anti-dog IgG
the treatment ended (ie, 24 months after enrollment). fluorescence-labeled secondary antibodiesn were used
Dogs that did not complete the study were also evalu- for the IFA procedures. Indirect immunofluorescence as-
ated and had PCR assay and IFA performed at their says were performed as previously described.36 Confir-
last follow-up visit if possible. Echocardiographic and matory ELISAs for T cruzi were performed as previously
ECG evaluations were performed for dogs that had described36 at Laboratorio de Enzimología de Parasitos,
relevant physical examination abnormalities when Universidad de Los Andes, Mérida, Venezuela.
deemed necessary by the attending veterinarian and
authorized by the client (for privately owned dogs). Other evaluations—Complete blood counts
Necropsy was performed on deceased dogs of the and serum biochemical analyses were performed
treatment group if possible. The study end points at various commercial veterinary laboratories or
were clinician-reported outcomes (including subjec- in house at various hospitals. Plasma was obtained
tively assessed improvement, persistence, or worsen- from peripheral blood samples according to hospital-
ing of clinical signs assessed from veterinary clinical specific protocols for measurement of itraconazole
observation) and indications of elimination of T cruzi concentrations in dogs of the treatment group as de-
from the peripheral blood, including negative results scribed. Samples were stored and shipped with ice
of PCR assay analysis, improvement in clinical signs packs under refrigeration at 1°C for measurement of
with or without seroconversion and normalization of itraconazole concentrations at a veterinary clinical
tissues on histologic examination, and differences in pharmacology laboratory.o
survival between treatment and control group dogs. Echocardiography was performed in primary
Dogs that had clinical improvement were defined as treatment locations by veterinarians aware of the
those that had improvement in ≥ 1 assessed physi- treatment group assignment of the dogs; the equip-
cal examination observation (eg, appetite, activity ment used varied by facility. Ventricular free wall,
level, or ascites) by the time of last follow-up. Death chamber size, and septal thickness measurements as
or euthanasia attributable to T cruzi infection was de- well as fractional shortening were recorded. A stan-
fined as either event associated with cardiomyopathy dard lead II ECG tracing was recorded by veterinary
caused by the disease, including death attributable to staff using an ECG unit for ≥ 5 minutes. Two-view
arrhythmia in a dog with this condition, euthanasia of right lateral and ventrodorsal thoracic radiographic

4 JAVMA | AUG 1, 2019 | VOL 255 | NO. 3

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views were also obtained in primary treatment loca- of the control group than for dogs of the treatment
tions when applicable. The echocardiography and group. The log-rank (Mantel-Cox) test was used to de-
ECG interpretations were read and summarized by 1 termine whether there was significant difference in
author (RM) and additionally classified as normal or the overall survival distributions of dogs that died of
abnormal for data analysis purposes. T cruzi infection between groups. All analyses were
Examination of heart tissue for dogs of the performed with statistical software.r
treatment group that died or were euthanized dur-
ing the study was performed at 1 of 2 pathology Results
laboratories.p,q Complete necropsy was performed
when possible. Heart tissue was examined by light One hundred twenty-one dogs were enrolled
microscopic evaluation of H&E-stained slides. Areas in the study (105 [16 government-owned and 89
involving the interventricular septum, apex, and privately owned] in the treatment group and 16 [4
both free walls of the heart were examined for in- government-owned and 12 privately owned] in the
flammation, fibrosis, and presence of amastigotes. control group). The initial enrollment included 125
dogs; however, 4 were excluded because they re-
Statistical analysis ceived compounded itraconazole. Twenty breeds and
The proportions of dogs that had clinical im- 1 mixed breed were represented, and the median age
provement by the end of the study and the propor- was 4.93 years (range, 5 days to 14 years). There were
tion of deaths attributable to T cruzi infection as well 63 males (27 castrated and 36 sexually intact) and
as baseline and 24-month (or last available) follow-up 58 females (33 spayed and 25 sexually intact). In the
clinical and biological results (frequencies of clini- control group, the number of dogs housed outdoors
cal signs attributable to T cruzi infection, normal vs versus indoors was the same (8/16 for each hous-
abnormal ECG and echocardiographic findings, posi- ing type), whereas in the treatment group, 69 of 105
tive and negative IFA results, and decreased antibody (66%) dogs were housed outdoors and 36 (34%) were
titers during the study period) were compared be- housed indoors.
tween the treatment and control groups. Results of Most (89/105 [85%]) dogs in the treatment group
PCR assays (positive vs negative) were compared be- had amiodarone administered according to the load-
tween groups at baseline and at each follow-up visit. ing dose protocol before receiving the maintenance
Analysis of clinical improvement by the 24-month or dose. The remaining 16 (15%) dogs in this group (all
last follow-up visit (yes vs no) included dogs that be- military working dogs) did not receive a loading dose
gan the study with clinical signs and dogs that devel- prior to the maintenance dose. Three dogs received
oped such signs during the study; findings at the last itraconazole at approximately 10 mg/kg, PO, every
available follow-up were used for dogs that died or 48 hours (instead of every 24 hours) because of low
were euthanized before the end of the study. Given body weight.
the small sample size of the control group, we used Eighty-six (82%) dogs in the treatment group
nonparametric comparisons, with Fisher exact and χ2 completed the study, and 19 (18%) died before the
tests for tabular analysis as appropriate for expected 24-month follow-up. The mean treatment period for
cell sizes and 2-sample Wilcoxon rank sum (Mann- dogs of the treatment group that did not complete
Whitney) tests for continuous variables. Repeated- the study was 138 days (median, 145 days; range, 30
measures MANCOVAs were performed to determine to 280 days). Eight of 16 dogs in the control group
the impact of combined amiodarone and itraconazole finished the study; 7 died, and 1 was lost to follow-up
treatment on multiple dependent variables (ECG [ab- 60 days after starting the study. The study completion
normal vs normal result], IFA [positive vs negative rates for the 2 groups were similar (P = 0.47). The
results and antibody titers], and echocardiography proportion of dogs that died was significantly (P <
[abnormal vs normal result]) measured at multiple 0.001) lower in the treatment group than in the con-
time points. The analysis included between-group trol group (19/105 vs 7/15).
and within-group effects. Only the ECG, IFA, and Five of 105 (5%) dogs in the treatment group had
echocardiography data were included as dependent deaths attributed to T cruzi infection by the 24-month
variables in this analysis; age, sex, breed, and housing follow-up; 1 was euthanized, and 4 acute deaths oc-
type (indoor vs outdoor) of the dogs were covariates curred in a kennel or at home during the treatment
controlled for in these analyses. Normal distribution period. In contrast, 6 of 15 dogs in the control group
of antibody titer data was confirmed by use of the had deaths attributed to the disease during the same
Shapiro-Wilk test. Values of P < 0.05 were considered period; 2 were euthanized and 4 died acutely. The
significant for all analyses. intergroup difference in rates of death attributable to
Kaplan-Meier survival analysis was conducted to the disease was significant (P = 0.003).
determine survival distributions of the dogs that died At the start of the study, the proportions of dogs
of T cruzi infection in each group. The duration of ob- with the most commonly reported clinical signs
servation to event (death) was 0 to 24 months, which (lethargy, ascites, hyporexia or anorexia, and exces-
was the duration of the study. This analysis was per- sive panting or coughing) did not differ between the
formed to determine whether the incidence of death treatment and control groups (Table 1). However,
attributed to T cruzi infection was greater for dogs with the exception of ascites, the proportions of dogs

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Table 1—Presence of common clinical signs attributed to Trypanosoma cruzi infection (American trypanosomiasis [commonly
called Chagas disease]) for 121 naturally infected dogs in a study to evaluate the clinical, serologic, parasitological, and histopatho-
logic outcomes following treatment with amiodarone and itraconazole.
Baseline Last follow-up
Treatment Control Treatment Control
Variable (n = 105) (n = 16) P value (n = 105) (n = 15) P value
Lethargy 28 (27) 1 (6) 0.116 2 (2) 6 (40) < 0.001
Ascites 10 (10) 0 (0) 0.35 4 (4) 1 (7) 0.49
Hyporexia or anorexia 21 (20) 1 (6) 0.29 3 (3) 5 (33) 0.001
Excessive panting or coughing 12 (11) 0 (0) 0.36 0 (0) 5 (33) < 0.001
Dogs of the treatment group received amiodarone hydrochloride and itraconazole for 12 months. Dogs of the control group did not receive
antitrypanosomal medications. Last follow-up data represent findings for dogs 24 months after the start of the study (n = 86 in the treatment group
and 8 in the control group) or at the last follow-up visit for dogs that did not survive to the 24-month time point (19 and 7 in the treatment and
control groups, respectively). Data are shown as number (%) of dogs in the group; some dogs had multiple clinical signs. One dog in the control
group was lost to follow-up during the study period. The P value reflects comparison between groups for the time point as determined by the χ2
or Fisher exact test; values of P < 0.05 were considered significant.
See Figure 1 for drug administration details.

that had these clinical signs were significantly greater Parasitemia assessment by PCR assay
for the control group than for the treatment group by Proportions of dogs in the treatment and control
the end of the study. groups that had positive PCR assay results for T cruzi
Fifty-four of 105 (51%) dogs in the treatment in blood at the start of the study (baseline) were com-
group had clinical signs attributed to T cruzi infec- parable (P = 0.754; Table 2). For the treatment group,
tion at the initial evaluation, and only 1 of 54 (2%) the positive result rate was 0% for the 93 tested dogs
failed to show improvement or resolution of clinical at the 6-month visit and 0% for the 90 tested dogs at
signs. For the control group, 1 of 16 dogs had clinical the 9-month visit (both significantly [P < 0.001] lower
signs at the start of the study, and 10 had clinical signs rates, compared with the baseline value); similar re-
consistent with disease progression at the end of the sults were found for PCR assay results at the 12-month
study. By the end of the 24-month follow-up period visit (time of final treatment) and the 24-month fol-
(or the last evaluation for dogs that did not survive to low-up (1% of 87 and 0% of 86 tested dogs, respec-
24 months), a significantly (P < 0.001) greater propor- tively). The positive result rates for control dogs were
tion of dogs in the treatment group had improvement 73%, 67%, and 67% for 15 tested dogs at the 6-, 9-, and
in ≥ 1 clinical sign (lethargy, hyporexia or anorexia, 12-month visits. At the 24-month follow-up, 4 of the 8
excessive panting, and ascites; 53/54 [98%]), com- known surviving control group dogs had positive re-
pared with the control group (0/10 [0%]). sults. The prevalence of parasitemia as determined by
PCR assay was significantly (P < 0.001) greater for the
Adverse events control group than for the treatment group at every
Fifteen of 105 (14%) dogs in the treatment evaluation after baseline.
group had adverse events attributable to the treat-
ments or treatment combination. Recorded events Serologic findings
included lethargy (n = 2), gastrointestinal signs Nineteen of 84 (23%) treatment group dogs and
(inappetence, vomiting, and diarrhea [alone or in 0 of 8 control group dogs that initially had positive
combination [7]), weight loss (3), cutaneous erup- results by IFA for anti–T cruzi antibodies had sero-
tions (2), and serum concentrations of alanine converted as assessed with this method by the time
transaminase above the reference range (10). Elev- of the last follow-up examination. The proportion of
en of these 15 dogs had high plasma concentrations dogs with a decrease in IFA-measured antibody titers
of itraconazole (≥ 3 µg/mL). The itraconazole dose over time was significantly (P = 0.02) greater for the
was reduced by 33% to 50% to achieve plasma con- treatment group (65/84 [77%]; mean titer at baseline
centrations in the range of 1 to 2 µg/mL for these and last follow-up, 2,282 and 893, respectively) than
dogs, and no further adverse events occurred fol- for the control group (3/8; mean titer at baseline
lowing dose adjustment. Halving the amiodarone and last follow-up, 1,704 and 3,349, respectively). No
dose ameliorated derangements in liver function treated dogs had a net increase in titer; however, 3
tests, clinical signs of lethargy and inappetence, of the 8 control dogs had a net increase in titer and 2
or both in the remaining 4 dogs. All dogs tested maintained a steady titer.
had measurable plasma concentrations of itracon- At baseline, mean anti–T cruzi antibody titers
azole that confirmed systemic exposure. Overall, were not significantly (P = 0.268) different between
amiodarone and itraconazole were well tolerated. the treatment and control groups (Table 3). At the
All clinically relevant treatment-related effects de- last follow-up, the mean antibody titer for the treat-
tected on physical examination were mild and re- ment group was significantly (P < 0.001) lower than
versible with dose modification. that for the control group. The mean change in titers

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Table 2—Results of PCR assays for T cruzi DNA in peripheral blood samples from the same dogs as in Table 1.
PCR assay result Treatment (n = 105) Control (n = 16) Total (n = 121) P value
Baseline 0.754
Negative 23/105 (22) 4/16 (25) 27/121 (22)
Positive 82/105 (78) 12/16 (75) 94/121 (78)
6 months < 0.001
Negative 93/93 (100) 4/15 (27) 97/108 (90)
Positive 0/93 (0) 11/15 (73) 11/108 (10)
9 months < 0.001
Negative 90/90 (100) 5/15 (33) 95/105 (90)
Positive 0/90 (0) 10/15 (67) 10/105 (10)
12 months < 0.001
Negative 86/87 (99) 5/15 (33) 91/102 (89)
Positive 1/87 (1) 10/15 (67) 11/102 (11)
24 months < 0.001
Negative 86/86 (100) 4/8 (50) 90/94 (96)
Positive 0/86 (0) 4/8 (50) 4/94 (4)
Changes in the number of dogs tested over time reflect the death of dogs (n = 26) or loss to follow-up (1) during the 24-month study period.
See Table 1 for remainder of key.

Table 3—Summary statistics for (reciprocal) anti–T cruzi antibody titers at baseline and at the last follow-up visit for the same
dogs as in Table 1.
Treatment group Control group
Variable Mean ± SD Median (range) Mean ± SD Median (range) P value
Baseline titer 2,282 ± 6,336 160 (20–32,768) 1,704 ± 2,713 640 (80–8,192) 0.268
Last follow-up titer 893 ± 2,424 80 (0–16,384) 3,349 ± 5,111 1,280 (160–16,384) < 0.001
Change in titers* –1,389 ± 5,436 160 (15,360–28,672) 1,645 ± 2,398 0 (6,144–8,192) 0.006
At baseline, there were 105 and 16 dogs in the treatment and control groups, respectively. Last follow-up data represent the titer recorded
24 months after the start of the study (n = 86 in the treatment group and 8 in the control group) or the last titer on record for dogs that did not
survive to the 24-month time point (13 and 1 in the treatment and control groups, respectively). The P values represent the comparison of titers
between groups by Wilcoxon rank sum test.
*Change in titer values represent the mean and median results for changes in individual dogs with data at both time points.

was a substantial decrease for the treatment group The proportions of dogs in the 2 study groups with
(mean ± SD change for individual dogs, –1,389 ± 5,436) normal ECG results at baseline were comparable (P =
and a substantial increase for the control group (1,645 0.705; Table 4). At the last follow-up evaluation, a signifi-
± 2,398); this difference was significant (P = 0.006) be- cantly (P < 0.001) lower proportion of control dogs had
tween groups. However, results of repeated-measures normal findings, compared with results for treatment
MANCOVA indicated that treatment did not have sig- group dogs. Abnormal ECG findings were detected in
nificant impact on the IFA results (positive vs negative; 25 of 92 treatment group dogs at baseline, and all these
P = 0.66) and only time period (last follow-up vs base- abnormalities resolved during the study period. For
line) was significantly (P = 0.02) associated with this the control group, 3 of 9 dogs had ECG abnormalities
outcome. The antibody titers for all dogs (the overall at baseline versus 6 of 9 (including the 3 with abnor-
study sample) at the last follow-up (mean ± SD, 893.70 ± malities noted at baseline) at the last follow-up. Results
2,808.81) were lower than those at baseline (2,282.481 of repeated-measures MANCOVA revealed that the
± 5,980.02); however, the time-by-treatment interac- combined amiodarone and itraconazole treatment was
tion was not significant (P = 0.98). significantly (P = 0.01) associated with the outcome of
normal ECG results (ie, sinus rhythm) at last follow-up
Electrocardiographic, echocardiographic, (found for 92/92 [100%] dogs in the treatment group vs
and radiographic findings 3/9 in the control group) and that the time-by-treatment
Electrocardiographic examinations were per- interaction also had a significant (P = 0.01) effect on
formed for 92 of 105 (88%) dogs in the treatment this variable. A higher proportion of normal ECG results
group and 9 of 16 dogs in the control group at the was found for dogs in the treatment group at the last fol-
start of the study, and all these dogs had ECG data low-up than at baseline (92/92 [100%] vs 67/92 [73%],
available for the last follow-up visit (Table 4). Echo- whereas the proportion of normal ECG results for dogs
cardiography was performed at baseline and repeated in the control group was lower at the last follow-up than
at the last follow-up visit for 25 and 21 dogs, respec- at baseline (3/9 vs 6/9).
tively, of the treatment group and for 6 and 4 dogs, The proportions of dogs with echocardiographic
respectively, of the control group. abnormalities did not differ between groups at base-

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Table 4—Results of ECG and echocardiographic evaluations at baseline and the last follow-up visit for the same dogs as in Table 1.
Variable Treatment Control Total P value
ECG findings at baseline 0.705
Normal 67/92 (73) 6/9 (67) 73/101 (72)
Abnormal 25/92 (27) 3/9 (33) 28/101 (28)
Second-degree AV block 1 0 1
Third-degree AV block 2 0 2
RBBB 2 0 2
Sinus tachycardia 2 2 4
PVC 11 1 12
PVC with sinus tachycardia 5 0 5
VT 2 0 2
Electrocardiogram findings at last follow-up < 0.001
Normal 92/92 (100) 3/9 (33) 95/101 (94)
Abnormal 0/92 (0) 6/9 (66) 6/101 (6)
ST segment depression 0 1 1
Sinus tachycardia 0 3 3
VT 0 2 2
Echocardiographic findings at baseline 0.750
Normal 9/25 (36) 2/6 (33) 11/31 (35)
Abnormal 16/25 (64) 4/6 (67) 20/31 (65)
Echocardiographic findings at last follow-up 0.570
Normal 15/21 (71) 0/4 (0) 17/25 (68)
Abnormal 6/21 (29) 4/4 (100) 8/25 (32)
Data represent the proportion (%) or number of dogs with a given finding per group. Some dogs had > 1 cardiac abnormality. Last follow-up
data represent findings 24 months after the start of the study (n = 74 treatment and 5 control group dogs for ECG and 14 treatment and 2 control
group dogs for echocardiography) or at the last follow-up visit for dogs that did not survive to 24 months (all other dogs that had these evaluations).
Within a time point, P values represent comparison of the proportions of dogs in each group that had normal (vs abnormal) results for a given
variable as determined by Fisher exact or c2 test.
AV = Atrioventricular. PVC = Premature ventricular contractions. RBBB = Right bundle branch block. VT = Ventricular tachycardia.

line (P = 0.750) or at the last follow-up (P = 0.570; included right-sided heart enlargement and mild to
Table 4). Abnormalities found in 16 of 25 treatment moderate generalized cardiomegaly. One of 5 control
group dogs included decreased fractional shortening dogs in which radiography was performed at base-
(n = 4), septal wall hypokinesis (1), left (3) and right line had generalized cardiomegaly with right and left
(7) atrial enlargement, mitral valve insufficiency (5), atrial and ventricular dilation. Follow-up radiography
tricuspid valve insufficiency (2), pulmonic valve in- was infrequently performed.
sufficiency (6), ventricular wall thickening with de-
creased chamber size (2) and pericardial effusion (2). Necropsy findings and reported causes
All treatment group dogs that had echocardiographic of death
abnormalities at baseline had improvement in these Necropsy was performed for 14 of 19 dogs in the
signs by the last follow-up, except for 1 dog that died treatment group that died during the study. Gross
of severe acute myocarditis and heart failure 16 days necropsy and histopathologic findings in these 14
after diagnosis. Valvular regurgitation was eliminated dogs included mild to severe myocarditis (n = 5); car-
in 11 of 13 dogs. However, 6 of 21 dogs evaluated at diac fibrosis, fatty infiltration, or both (6); nephritis
the last follow-up still had abnormalities present. Ab- (3); alveolar histiocytosis (2); lymphoid hyperpla-
normalities in 4 of 6 control group dogs at baseline sia of the spleen, lymph nodes, intestines, or tonsils
included moderate pulmonary valve insufficiency (n (alone or in combination; 3); chronic passive liver
= 1), mild mitral valve insufficiency (1), and moder- congestion (2); gastric periganglitis (1); and cardio-
ate bilateral atrial and ventricular enlargement (2). At megaly (8). Although none of the 14 dogs that un-
the last follow-up, findings for the latter 2 dogs had derwent necropsy completed the full 24 months of
progressed to severe bilateral atrial and ventricular the study, 9 were free of cardiac inflammation and
enlargement, and findings for the 2 dogs with valvu- had no amastigotes identified at necropsy. Prior to
lar insufficiency had remained the same. Results of death, 12 of these 14 dogs had clinical improvement.
repeated-measures MANCOVA revealed that treat- Five deaths (4 acute deaths and 1 euthanasia) were
ment did not have significant impact on the echo- attributed to cardiac complications of T cruzi infec-
cardiography results (normal vs abnormal; P = 0.17); tion; causes of death or euthanasia for the remaining
echocardiography results did not differ significantly 14 treatment group dogs included neoplasia (n = 3),
(P = 0.76) over time, and the time-by-treatment inter- concerns about zoonotic transmission of T cruzi (2),
action was not significant (P = 0.25) for this variable. intractable seizures (2), persistent ascites (1), acute
Forty-two dogs in the treatment group under- renal failure (2), megaesophagus (1), chronic spinal
went radiographic examination at baseline, and 17 cord degeneration (2), and limb fracture (1). One of
had signs of cardiomegaly. The signs were subtle and these dogs was euthanized after premature discon-

8 JAVMA | AUG 1, 2019 | VOL 255 | NO. 3

 Small Animals

val, 22.40 to 23.97 months), whereas that for dogs in

the control group was 15.64 ± 2.44 months (95% con-
fidence interval, 10.86 to 20.42 months). The differ-
ence in the survival distributions for treatment group
versus control group dogs is depicted in the Kaplan-
Meier survival analysis plot (Figure 1).

Discussion
Preliminary evidence from this small, 2-year
study that included 121 dogs with naturally occurring
T cruzi infection (105 dogs that received a combina-
tion of amiodarone and itraconazole for 12 months
[treatment group] and 16 dogs that received no
antitrypanosomal treatment [control group]) suggest-
ed that the study treatment was efficacious. Results
of baseline clinical and parasitological assessments
were comparable between the 2 study groups, and
several outcomes of interest, including the main end
points of improvement in clinical signs, negative re-
sults of PCR assay for T cruzi DNA, and survival until
death attributable to T cruzi infection, differed signif-
Figure 1—Kaplan-Meier plot of cumulative survival over icantly between groups, with more favorable results
time until death attributable to naturally acquired Trypano- found for dogs in the treatment group.
soma cruzi infection for 121 dogs in a study to assess clinical,
serologic, parasitological, and histopathologic outcomes fol- Clinically, cardiomyopathy is the most promi-
lowing treatment with amiodarone and itraconazole. Dogs of nent manifestation of chronic T cruzi infection in
the treatment group (dashed line; n = 105) were treated with dogs in North America, with very few reports13,14,19 of
a combination of amiodarone hydrochloride and itraconazole gastrointestinal or neurologic involvement. Chronic
for 12 months. Amiodarone was given as a maintenance dos-
age of 7.5 mg/kg [3.4 mg/lb], PO, q 24 h or a loading dosage cardiomyopathy can occur months or years after the
of 15 mg/kg, PO, q 12 h for 7 days followed by 15 mg/kg [6.8 initial infection but usually develops within 8 to 36
mg/lb], PO, q 24 h for 14 days and then the described mainte- months after inoculation.13,14,19 As in human patients,
nance dosage; the treatment was supplied as scored 200-mg diffuse myocarditis and fibrosis lead to a number of
tablets, and the calculated dose was rounded to the nearest conduction disturbances,17,40 along with clinical signs
50 mg. Itraconazole was given at a dosage of 10 mg/kg [4.5
mg/lb], PO, q 24 h; the treatment was supplied as 100-mg of right-sided or left-sided heart failure, such as dys-
capsules, and the calculated dose was rounded down to the pnea (because of pulmonary edema), ascites, and
nearest whole capsule (for patients with low body weight, hepatomegaly.14 Dogs that enter the chronic phase of
the protocol was revised to deliver approx 10 mg/kg, q 48 the disease without overt clinical signs (commonly
h). Itraconazole dosages were adjusted after measurement
at steady state to maintain a plasma concentration of 1 to termed the chronic asymptomatic phase) may not
2 µg/mL. Dogs of the control group (solid line; n = 16) did develop clinical signs; however, ventricular arrhyth-
not receive antitrypanosomal medications. At the end of the mias may be induced by exercise, potentially leading
24-month study period, 86 dogs of the treatment group and 8 to death.14,19 Our results supported these findings; the
dogs of the control group were alive (and 1 dog of the control most commonly identified ECG abnormalities were
group had been lost to follow-up); 5 of 105 treatment group
and 6 of 15 control group dogs had deaths attributable to T premature ventricular contractions, followed by ven-
cruzi infection. Vertical marks indicate censored data points. tricular tachycardia, sinus tachycardia, atrioventricu-
lar block, and right bundle branch block.
The combination of amiodarone and itraconazole
tinuation of the treatment protocol and was included used in the present study was selected to target spe-
in the category of other causes of death (ascites). Of cific metabolic pathways of the parasite. Amiodarone
the 7 control group dogs that died during the study, is an antiarrhythmic agent that is frequently pre-
the only death that was not attributed to T cruzi in- scribed to prevent complex arrhythmias in human
fection was attributed to lymphosarcoma; these dogs patients with cardiomyopathy due to T cruzi infec-
did not undergo necropsy as part of the study. tion36 and was used in the present study not only in
an effort to prevent development of exercise-induced
Survival times for dogs with death and potentially lethal ventricular arrhythmias but
attributed to T cruzi infection also because of its direct activity against T cruzi.39,40
There was a significant (P < 0.001) difference be- This agent disrupts calcium homeostasis in the para-
tween the treatment and control groups in survival site by inducing release of calcium from intracellular
distributions of dogs with death attributed to T cruzi stores (such as the giant mitochondrion and acidocal-
infection during the 24-month study period. Mean ± cisomes, which are unique organelles involved in the
SE estimated survival time for dogs in the treatment bioenergetics of these organisms).39 Amiodarone also
group was 23.19 ± 0.4 months (95% confidence inter- blocks biosynthesis of sterols,39,42 which is lethal in

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 Small Animals

T cruzi because the parasite uses these sterols instead crease in the group mean to less than half of the base-
of cholesterol to synthesize the ergosterol needed for line value) versus control dogs were nonsignificant
proliferation.25,47 Moreover, the spectrum of action of in this analysis. A possible explanation for this is the
amiodarone extends beyond its antiparasitic effect tendency for T cruzi–specific antibodies to persist in
to include direct modulation of calcium homeostasis the bloodstream for many years, even after treatment
in cardiomyocytes. The arrhythmogenic events that for chronic Chagas disease in people.49 Despite the
result from T cruzi infection have been shown to de- prolonged period of treatment, only a few animals
pend on the ability of the parasite to disrupt commu- had adverse effects attributable to itraconazole or
nication through gap junctions by decreasing concen- amiodarone treatment, all of which were reversible
trations of connexin 43 (an essential gap protein) to when the dosage was reduced.
interact with actin filaments.48 It was also confirmed Importantly, because of the intracellular nature
experimentally that amiodarone-treated cardiomyo- of T cruzi and its well-known capacity to reside in so-
cytes recover their normal distribution of connexin called sanctuary sites such as adipose tissue,50 mono-
43 and spontaneous contractility.48 We believe that nuclear phagocytic cells,51 and cardiomyocytes,52
the complete reversal of arrhythmias observed in our delivery of treatment poses a challenge. Neverthe-
study, with resolution of the described ECG abnor- less, itraconazole concentrations in cardiomyocytes,
malities in 25 of 25 tested dogs of the treatment group adipocytes, and splenic tissue have been found to be
and a significant association between treatment and higher than those in plasma in people and in labora-
the outcome of normal sinus rhythm on ECG at the tory animals, indicating that parasites are exposed to
last follow-up visit (as assessed by repeated-measures higher concentrations of the drug in these tissues.52,53
MANCOVA), suggested an underlying amiodarone- Amiodarone and itraconazole also accumulate in
induced recovery of cellular events to yield clinical macrophages.53–55
effects. Other possible explanations for the elimina- Amiodarone and itraconazole concentrations are
tion of arrhythmias included decreased myocardial 10 and 5 times as high, respectively, in myocardium
inflammation and scarring, decreased amounts of as in plasma, indicating that both drugs accumulate at
proinflammatory cytokines being released from in- the primary target organ for T cruzi.54,56,57 Fat is also
flamed or infected tissue, and improved diastolic fill- a reservoir from which infection can be reactivated,
ing resulting in better coronary perfusion and deliv- and therefore, penetration of the adipose tissue is
ery of oxygen to the myocardium. critical for elimination of the chronic phase of infec-
In addition, we observed improvement in some tion.58 Both of the drugs used in the present study are
of the echocardiographic variables for treated dogs highly lipophilic, with concentrations of itraconazole
of our study, such as increased fractional shorten- in adipose tissue 25 times those in plasma.59 Further-
ing, elimination of valvular regurgitation, reduction more, both agents have very long half-lives in dogs
of ventricular wall thickening, and enhanced septal (amiodarone, 3.2 days; itraconazole, 2.1 days) 60,61 and
wall kinesis, which suggested improved myocardial are relatively safe with dose-dependent and reversible
function in these dogs. This finding could be attrib- toxic effects.
utable to the way in which amiodarone promotes In contrast, benznidazole, which is the drug most
recovery of cardiac cells by recovery of the fibrillar widely used for treatment of patients infected with
organization of F-actin, consistent with the observa- T cruzi, has poor tissue penetration35 and does not
tions of Adesse et al48 in regard to the finding that re- accumulate in macrophages.62 Moreover, benznida-
duced contractility of cardiomyocytes resulting from zole has several limitations with regard to its efficacy
T cruzi infection is corrected after treatment with against this organism in the acute and chronic phases
amiodarone to a degree that is indistinguishable from of infection in dogs. Benznidazole has an unfavor-
that in noninfected healthy cardiomyocytes.48 able pharmacokinetic profile that includes a short
Itraconazole was administered concomitantly elimination half-life of only 13 hours and a maximum
with amiodarone in this study in an effort to take plasma concentration of 2.4 µg/mL, which makes it
advantage of the known synergism between the 2 difficult or impossible to maintain the recommended
drugs.36 Like other azoles, itraconazole is known trypanocidal concentration of 3 to 6 µg/mL.63 Benz-
to act against T cruzi by inhibition of cytochrome nidazole reaches concentrations in target tissues that
P450-dependent C14α-sterol demethylase, which is are much lower than those in plasma, with reported
involved in several membrane-bound enzymatic path- concentrations in the heart and spleen that are 32%
ways.24,36 Although imidazole has previously been re- and 27%, respectively, of concentrations achieved in
ported to be ineffective in the treatment of T cruzi plasma; these sites receive an amount of the drug that
infection in dogs,13 the present study yielded some is far below the minimum inhibitory concentration
interesting results. values, and the strain of T cruzi also impacts the ef-
Although the seroconversion rate to negative ficacy of benznidazole.35,64,65 The T cruzi DTU I strain
results for treatment group dogs that initially tested is the only strain that has been shown to infect peo-
positive for anti–T cruzi antibodies by IFA was 19 of ple autochthonously in the United States66 and is a
84 (23%), MANCOVA results did not reveal this as sig- major variant that causes American trypanosomiasis
nificantly associated with treatments, and the lower in dogs in the United States.67 This strain is inherent-
posttreatment antibody titers in treated dogs (a de- ly resistant to benznidazole, which failed to resolve

10 JAVMA | AUG 1, 2019 | VOL 255 | NO. 3

 Small Animals

parasitemia in 4 of 4 acutely experimentally infected mean age of 4.5 years have been reported to survive
dogs treated for 45 days after inoculation with the T for a maximum of 5 months after diagnosis of T cruzi
cruzi DTU I strain.68 Other limitations include poor infection.21
drug availability in the US market and toxic effects Acute-phase manifestations of T cruzi infection
for a total dose of 18 g reported in the human medi- in dogs are usually missed because of the subtle de-
cal literature,69 although most dogs seem to tolerate gree and short duration of signs, such as anorexia for
a 45- to 60-day treatment regimen fairly well.70 As- 1 to 2 days or acute self-limiting diarrhea, so most
sessment of therapeutic effectiveness of a treatment cases are not diagnosed until clinical signs are ob-
for T cruzi can be challenging because the disease served in the chronic stage unless detected inciden-
develops slowly and because anti–T cruzi antibod- tally during the chronic phase without overt clinical
ies persist in a large percentage of patients for many signs by routine serologic screening.13,14 Historically,
years after treatment. Although some authors have specific antiparasitic treatment was likely to be inef-
reported a decrease in specific antibodies following fective during the chronic stage because of fibrosis
treatment and suggested that this could be used as and cardiac damage, and treatment was mainly sup-
an indicator of cure, different serologic profiles are portive and directed toward management of cardiac
detected with ELISA, IFA, and Western blotting in signs.13,36,40 The clinical relevance of our findings was
chronically infected treated dogs, which indicates that successful treatment was achieved by exploiting
these methods alone are not optimal. The expensive the synergistic effects of amiodarone and itracon-
and lengthy patient follow-up required with these azole, suggesting that this combined drug treatment
methods places an unrealistic burden on clinicians has efficacy against T cruzi in dogs with naturally ac-
and clients. However, PCR assays have high sensitiv- quired disease.
ity for detection of circulating trypomastigotes and, A limitation of the present study was that identi-
despite the intermittent shedding of the organism in fication of the T cruzi strains by DTU identification
peripheral blood, have been shown to be useful for was not performed in the study dogs. This is impor-
monitoring clearance of the parasite.68 In our study, tant, given that the DTU classifications have clinico-
we used serial PCR assay evaluations with multiple pathologic implications with regard to the biological
targets to increase our ability to detect the organism features and progression of disease as well as drug
during and after treatment. Even though PCR assay susceptibility.32,73 Other limitations include the small
is not historically considered a reliable indicator of size of the control group, lack of ECG and echocar-
cure because of its low sensitivity for detection of T diography data for all dogs, lack of blinding during
cruzi in patients with chronic infection,71 the finding clinical evaluations and assessment of ECG and echo-
of negative PCR assay results in nearly all tested dogs cardiography data, and inability to evaluate success
of the treatment group beginning 6 months after rates on the basis of disease phase in the dogs.
treatment was initiated (with a single positive test at Treatment of T cruzi–infected dogs that do not
the 12-month time point) supported that a cure was have signs of disease may have an important part to
achieved in most dogs. Moreover, the value of persis- play in public health by reducing the risk of poten-
tent negative PCR assay results in monitoring of treat- tial transmission through breeding66 or exposure of
ment has been suggested by other investigators.72 infected animals to feeding vectors that can transmit
Our investigation of clinical outcomes revealed the disease to other dogs. Reduction of parasitemia
that the proportion of dogs in the treatment group inhibits the spread of the disease by eliminating try-
with deaths attributable to T cruzi before the end of pomastigotes available for a blood meal by a triato-
the study was significantly less than that of the con- mine insect and could reduce the risk of zoonotic
trol group dogs (5/105 [4.8%] vs 6/15 [40%]). Survival transmission, especially in endemic areas. Because
distributions of dogs with death attributed to T cruzi T cruzi infection in animals is not a reportable condi-
infection differed significantly between the treatment tion in Texas or in other southern parts of the United
and control groups, with mean survival times of 23.19 States,9,74 the number of reported cases is likely a sub-
and 15.64 months, respectively. Furthermore, improve- stantial underestimate of the true incidence. The coex-
ment in ≥ 1 clinical sign by the last follow-up visit was istence of important disease vectors, such as Triatoma
significantly more common for dogs of the treatment sanguisuga, Triatoma gerstaeckeri, and Triatoma
group (53/54 [98%]) than for dogs of the control group lecticularia,74–76 and the variety of susceptible mam-
(0/10). One of the most profound results was the previ- malian reservoirs9,74 in a broad spectrum of ecological
ously mentioned significant difference in ECG results regions could favor occurrence of this disease at epi-
over time. Conduction abnormalities in 25 of 25 (100%) demic proportions in the southern United States.
treatment group dogs had resolved by the time of last Veterinarians and pet owners should be aware
follow-up, whereas 3 of 9 control group dogs had no of the potential threat of T cruzi infection in domes-
improvement in this variable and 3 additional dogs in tic dogs and be familiar with the clinical signs of the
this group developed conduction abnormalities by the disease as well as the emerging alternatives to treat
last follow-up, supporting that the combined amioda- it. The present study represented an effective inter-
rone and itraconazole treatment improved the cardiac national collaborative effort between physicians, vet-
status of treated dogs. These findings were important, erinarians, and biologists and was an example of the
given that untreated chronically infected dogs with a One Health Initiative supporting the vision of improv-

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ing animal and human health globally through such 12. AVMA. US pet ownership & demographics sourcebook. Scha-
collaboration to address critical needs.77 umburg, Ill: AVMA, 2012.
13. Barr SC. Canine Chagas’ disease (American Trypanosomia-
sis) in North America. Vet Clin North Am Small Anim Pract
Acknowledgments 2009;39:1055–1064.
14. Barr SC, Gossett KA, Klei TR. Clinical, clinicopathologic, and
No third-party funding or support was received in connec-
parasitologic observations of trypanosomiasis in dogs infect-
tion with this study or the writing or publication of the manu-
ed with North American Trypanosoma cruzi isolates. Am J
script. The authors declare that there were no conflicts of interest.
Vet Res 1991;52:954–960.
The authors thank Dawn Boothe, Clinical Pharmacology
15. Berger SL, Palmer RH, Hodges CC, et al. Neurologic mani-
Laboratory, Auburn University College of Veterinary Medicine for
festations of trypanosomiasis in a dog. J Am Vet Med Assoc
technical expertise.
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