Introduction
Chagas disease is predominately characterized by cardiac abnormalities in dogs that develop myocarditis and myocardial fibrosis from Trypanosoma cruzi infection.1–6 In most cases, the time frame in which a dog has become infected is unknown, and the cardiac damage that develops because of infection leads to variable findings. While some infected dogs do not have any detectable cardiac abnormalities, others will have cardiac chamber enlargement, ventricular dysfunction, increased cardiac troponin I (cTnI) concentration, and ECG abnormalities characterized by abnormal QRS morphology, conduction system abnormalities, and ventricular and supraventricular arrhythmias.7–13 Infected dogs can also develop lethargy, pale mucous membranes, prolonged capillary refill time, generalized lymphadenopathy, splenomegaly, and hepatomegaly.3
In experimentally infected dogs, ECG abnormalities detected in the acute stage of infection include abnormal ECG complex morphology characterized by decreased R wave amplitude and conduction system abnormalities characterized by atrioventricular (AV) and bundle branch block.2 Ventricular arrhythmias have been observed as early as 2 months after infection and echocardiographic abnormalities at > 6 months after infection.2 In studies of naturally infected dogs, similar ECG abnormalities as well as variable degrees of echocardiographic chamber enlargement with ventricular dysfunction and elevated cTnI concentrations have been documented.14–16
The chronic asymptomatic stage of Chagas disease is characterized by positive serology and an absence of clinical signs, although ECG abnormalities may be detected during this time and sudden death can occur.2,6 In the chronic asymptomatic stage of disease in humans, routine monitoring with ECG and echocardiography aids in detecting cardiac disease.7,17 In government working dogs at the Texas-Mexico border that were evaluated with ambulatory ECG (Holter), abnormalities were detected more often in serologically positive (76.5%) and discordant (100%) dogs compared to negative dogs (11.1%).13 Additionally, serum cTnI concentrations were higher in seropositive (SP) dogs. Ambulatory ECG monitoring, comprehensive echocardiographic studies, and cardiac MRI have been used to detect cardiac disease from T cruzi infections; however, these approaches require specialized equipment, time to perform, and expertise to interpret.13,18 While ambulatory ECG has a higher likelihood of detecting intermittent arrhythmias compared to a standard ECG,8 conduction abnormalities and altered complexes (prolonged intervals, small R waves, splintering of the QRS complex) would be readily detected by standard ECG, are some of the earliest changes identified in experimentally infected dogs, and are present in naturally infected dogs and humans.2,8,19
In humans, recommendations for the diagnosis of Chagas disease include identifying risk factors to guide who should be screened and which diagnostic tests should be performed; however, a lack of available diagnostics and treatments can lead to underdiagnosis and healthcare concerns.17,20 Studies have shown a high prevalence of T cruzi infection in dogs in some areas of the southern US with high transmission pressure and the presence of SP dogs throughout the US due to animal transport.21–25 Additionally, there is a need for recommendations for screening and routine monitoring of infection in military working dogs.25,26 These factors, coupled with the potential for limited routine access to advanced diagnostic techniques,18 suggest it would be useful to investigate a simplified set of diagnostic tests for evaluating Chagas disease in dogs that would be readily accessible in practice, similar to what is proposed for infected humans.17
The objective of this study was to determine whether T cruzi infection serostatus was associated with cardiac abnormalities by use of a simplified diagnostic evaluation in dogs that live in high-risk environments for T cruzi infection.
Methods
Study population
A convenience sample of client-owned dogs residing in 4 multidog kennels in a high-risk area for T cruzi infection in South Texas were enrolled in a prospective, cross-sectional study conducted in September 2022. The dogs were primarily trained for sporting activities and were monitored and cared for daily by kennel owners and staff. The kennel environments have partial or complete exposure to the outdoors, and environmental insecticides are utilized. All dogs were considered to be in good health with no significant medical history or concurrent diseases noted and were fed traditional diets. All dogs received routine vaccinations as well as medication for heartworm prophylaxis, intestinal parasite control, and protection from fleas and ticks. Dogs were excluded if they were not able to have diagnostics performed on the study date or if they had received antiprotozoal or antiarrhythmic medications including benznidazole, itraconazole, or amiodarone at any point prior to enrollment. For each dog enrolled, demographic data, history, and a functional evaluation of cardiac health (FETCH) questionnaire score were recorded27 and an ECG and echocardiogram were performed. Blood samples were obtained, processed, and stored for evaluation of serum cTnI concentration, 2 serologic tests for T cruzi, and PCR. The study was approved by the IACUC (AUP 2021-0131), and informed consent was obtained for each dog.
Laboratory analysis
At the time of evaluation, a minimum of 6 mL of blood was collected from a jugular vein, divided into serum separator tubes, spun, separated, and stored at –20 °C until analysis was performed. Serologic tests included an indirect fluorescent antibody (IFA) for anti–T cruzi IgG antibodies submitted to the Texas A&M Veterinary Medical Diagnostic Laboratory, with titers ≥ 1:20 considered positive, and a rapid immunochromatographic assay for anti–T cruzi antibodies validated for humans (Chagas STAT-PAK; ChemBio Diagnostics), in which an absence of a band was considered negative, the development of a colored band was considered positive, and strength of the response was scored on a scale from 1 to 4. Real-time quantitative PCR for T cruzi DNA was performed on buffy coat from blood samples, with a cycle threshold value of < 35 cycles considered positive.21 Serum cTnI concentrations were measured with a high-sensitivity assay (ADVIA Centaur XP high-sensitivity troponin I assay; Siemens Healthcare Diagnostics Inc), with a reported range of 2.5 to 168 pg/mL that has been validated in dogs.28
Dogs were classified into 3 groups for analysis on the basis of serology results as follows: SP if positive results were obtained on both serology tests, seronegative (SN) if both results were negative, and serologically discordant (SD) if results were positive on only 1 of the 2 tests.
Electrocardiography
Electrocardiography recordings were obtained with a computer-aided ECG analysis program (CardioPet ECG device; Idexx Laboratories Inc) that is available to veterinary practitioners. The standard protocol included a 30-second recording, with an average heart rate calculated in beats per minute and complexes measured in lead II on a sinus beat. Parameters reported by the program included heart rate, P wave duration, PR interval duration, QRS interval duration, QT interval duration, P wave amplitude, and R wave amplitude. Additionally, the program provides a designation as normal or abnormal and notification of arrhythmias detected. All variables were reviewed by at least 1 of 2 authors (KAZ and ABS) to ensure accuracy, and adjustments were made to computer-aided measurements when appropriate. Measurements were evaluated by use of published reference ranges for dogs.29 Amplitude of the R wave < 0.5 mV and > 3 mV was considered abnormal. The ECG recordings were reviewed, and splintering of the QRS complex, supraventricular or ventricular arrhythmias, and second- or third-degree AV block were recorded if present. Dogs were classified by the investigators as having either the presence or absence of conduction abnormalities defined as prolonged duration of the P wave, PR interval, or QRS interval; splintering of the QRS complex; or identification of AV block of any degree.
Echocardiography
Transthoracic echocardiographic studies were performed by a board-certified cardiologist (ABS) or experienced veterinary sonographer (MH). Prior to performing the echocardiographic studies, the sonographer was provided with detailed instructions for image acquisition and measurements for this study. Echocardiographic examinations were brief, focusing on views obtained from the right side of the thorax and limited to 7 measured variables. The echocardiographic studies performed by the cardiologist were reviewed on a digital workstation (GE EchoPAC v203; GE HealthCare), and a single measurement was made and recorded by the same cardiologist (ABS). The echocardiographic studies performed by the sonographer were reviewed on a portable ultrasound unit (MyLab Omega; Esaote SPA), and a single measurement was made by the same sonographer and screen captured for review by the cardiologist. All images and measurements were reviewed by the cardiologist.
From a right parasternal short-axis view, the left ventricular internal dimensions at end-systole (LVIDsN) and left ventricular internal dimensions at end-diastole (LVIDdN) were measured from M-mode images and normalized to body weight. Measurements for LVIDdN and LVIDsN were considered outside the 95% CI when they measured > 1.85 and > 1.26, respectively.29 Fractional shortening (FS) of the left ventricle was calculated from M-mode measurements, and values < 20% were considered abnormal.30 M-mode images at the level of the mitral valve were used to measure E-point septal separation as previously described, and values > 6.5 mm were considered abnormal.31 The left atrium-to-aorta ratio (LA:Ao) was calculated from measurements of the LA and Ao, and values > 1.57 were considered abnormal.32 In a right parasternal long-axis 4-chamber view, the diameters of the right atrium (RA) and LA were measured 1 frame before mitral or tricuspid valve opening across the midsection of each chamber parallel to the valve annulus.33,34 An LA:RA ratio was calculated, and a ratio of < 1 was considered abnormal and evidence of RA enlargement. Right ventricular internal diameter in diastole was measured on the right parasternal long-axis 4-chamber view at the midventricle, perpendicular to the long axis of the ventricle, and was indexed to body weight, with values > 0.94 considered abnormal.35
Statistical analysis
Data were tabulated to examine for data-processing errors and for distribution shape where appropriate. Continuous variables were assessed for normality with the Shapiro-Wilk test. Data were not normally distributed and were compared with the Mann-Whitney U test for between–serostatus group comparisons or χ2 analysis for categorical data. Similar comparisons were made between dogs that were PCR positive and negative. Statistical significance was set at P < .05. Descriptive statistics are reported as median (range) or count (proportion), as appropriate. The relationship between evidence of T cruzi infection (ie, SP) and cardiac abnormalities was explored by use of the least absolute shrinkage and selection operator (lasso) to identify potentially important associations between serologic classification and dichotomous (ie, those designated normal or abnormal) explanatory variables. This process was duplicated while designating SD cases as either SP or SN. Association between these identified variables and serologic status were then tabulated and examined with χ2 tests. Analysis was performed with commercially available statistical software (Stata version 18, StataCorp; and Prism 7, GraphPad Software Inc).
Results
Of the 85 dogs initially considered available for evaluation, 32 were excluded for receiving prior medications and 7 were excluded because they did not have sufficient data or were not able to be fully evaluated on the study date. The remaining 46 dogs were purebred and represented by 10 breeds, with English Pointer (n = 16), English Cocker Spaniel (11), and Kelpie (7) being the most common. No other breed was represented by > 3 dogs. Median age was 5.0 years (range, 1.0 to 11.4 years) and weight was 21.7 kg (range, 9.6 to 58.0 kg). Sex distribution was equal (Table 1). All dogs scored 0 on the FETCH questionnaire, indicating the absence of clinical signs.
Demographic, laboratory, and cardiac diagnostic test results in 46 dogs at high risk for Trypanosoma cruzi infection and grouped by serostatus as positive, negative, or discordant on the basis of results of 2 serologic tests.
All dogs (n = 46) | Seropositive (n = 19) | Seronegative (n = 17) | Serologically discordant (n = 10) | |
---|---|---|---|---|
Signalment | ||||
Age (y) | 5.0 (1.0–11.4) | 6.0 (2.0–10.5)a | 4.0 (1.0–11.4) | 4.6 (1.0–11.0) |
Weight (kg) | 21.7 (9.6–58.0) | 23.0 (15.0–38.5) | 21.2 (9.6–58.0) | 19.9 (10.2–32.5) |
Sex (male/female) | 23/23 | 9/10 | 11/6 | 3/7 |
Laboratory analysis | ||||
IFA positive | 21 | 19 | 0 | 2 |
STAT-PAK positive | 27 | 19 | 0 | 8 |
PCR positive | 9 | 7 | 1 | 1 |
cTnI (pg/mL) | 89.88 (8.75–862.49) | 126.00 (13.94–862.49)b | 55.82 (8.75–334.76) | 99.32 (11.80–798.83) |
cTnI > 168 pg/mL | 6 | 4c | 1d | 1e |
ECG | ||||
Heart rate (beats/min) | 115 (64–181) | 110 (84–172) | 120 (75–159) | 116 (96–181) |
P wave amplitude (mV) | 0.22 (0.02–0.47) | 0.22 (0.02–0.47) | 0.22 (0.11–0.4) | 0.21 (0.13–0.3) |
P wave amplitude > 0.4 mV | 1 | 1 | 0 | 0 |
P wave duration (ms) | 35 (16–48) | 34 (16–48) | 35 (30–40) | 36 (28–46) |
P wave duration > 40 ms | 4 | 3 | 0 | 1 |
PR interval (ms) | 106 (74–220) | 110 (82–220) | 103 (74–130) | 106 (94–118) |
PR interval > 130 ms | 1 | 1 | 0 | 0 |
QRS duration (ms) | 44 (28–87) | 46 (28–87) | 42 (34–64) | 45 (40–58) |
QRS duration > 70 ms | 1 | 1 | 0 | 0 |
R wave amplitude (mV) | 2.38 (0.28–6.35) | 2.02 (0.28–6.35)b | 2.83 (1.51–5.54) | 2.32 (1.64–3.26) |
R wave amplitude < 0.5 mV | 1 | 1 | 0 | 0 |
R wave amplitude > 3 mV | 10 | 3 | 6 | 1 |
QT interval (ms) | 186 (159–234) | 193 (159–234)c | 182 (163–209) | 179 (162–194) |
QT interval > 240 ms | 0 | 0 | 0 | 0 |
Presence of ventricular arrhythmias | 4 | 3 | 0 | 1 |
Presence of a conduction abnormality | 7 | 6 | 0 | 1 |
Echocardiogram | ||||
LVIDdN | 1.63 (1.31–2.02) | 1.62 (1.35–1.95) | 1.66 (1.40–2.02) | 1.60 (1.31–1.84) |
LVIDdN > 1.85 | 5 | 2 | 3 | 0 |
LVIDsN | 1.08 (0.73–1.44) | 1.05 (0.75–1.32) | 1.11 (0.73–1.44) | 1.09 (0.93–1.21) |
LVIDsN > 1.26 | 7 | 3 | 4 | 0 |
FS (%) | 29.3 (15.7–51.8) | 30.6 (21.9–51.8) | 29.0 (15.7–47.0) | 27.1 (22.1–34.0) |
FS < 20% | 1 | 0 | 1 | 0 |
EPSS (mm) | 5.3 (2.1–10.5) | 4.8 (2.1–7.7) | 5.5 (2.3–9.0) | 6.2 (3.3–10.5) |
EPSS > 6.5 mm | 11 | 2 | 5 | 4 |
LA:Ao | 1.18 (0.74–1.54) | 1.2 (0.97–1.45) | 1.14 (0.74–1.34) | 1.16 (0.8–1.54) |
LA:Ao > 1.57 | 0 | 0 | 0 | 0 |
LA:RA | 1.29 (1.02–1.86) | 1.38 (1.16–1.86) | 1.25 (1.08–1.42) | 1.19 (1.02–1.42) |
LA:RA < 1 | 0 | 0 | 0 | 0 |
iRVIDd | 0.58 (0.39–1.04) | 0.56 (0.39–0.85) | 0.60 (0.43–1.04) | 0.57 (0.46–0.74) |
iRVIDd > 0.94 | 1 | 0 | 1 | 0 |
Presence of MMVD | 14 | 9 | 2 | 3 |
Data are reported as median (range) or number of dogs.
Ao = Aorta. cTnI = Cardiac troponin I. EPSS = E-point septal separation. FS = Fractional shortening. IFA = Immunofluorescent antibody. iRVIDd= Right ventricular internal diameter in diastole indexed to body weight. LA = Left atrium. LVIDdN = Left ventricular internal dimension in diastole normalized to body weight. LVIDsN = Left ventricular internal dimension in systole normalized to body weight. MMVD = Myxomatous mitral valve disease. RA = Right atrium.
aP < .05, compared to negative.
bP < .05, compared to discordant.
cOne dog was PCR positive.
dThis dog was PCR negative.
eThis dog was PCR positive.
Laboratory analysis
Of the 46 dogs included, 19 (41%) were SP, 17 (37%) were SN, and 10 (22%) were SD. Nine dogs were PCR positive (7 SP, 1 SD, 1 SN). There were not any differences between groups for any variables based on PCR status. For the 21 of 46 dogs (19 SP, 2 SD) with a positive IFA test result, titers ranged from 1:40 to > 1:1,280 (Table 2). Titers were ≤ 1:80 in 6 dogs (6 of 21 [29%]) and ≥ 1:160 in 15 dogs (15 of 21 [71%]). For the 27 of 46 dogs (19 SP, 8 SD) with a positive Chagas STAT-PAK, scores ranged from 1 to 4. Scores for SP dogs ranged from 1 to 4 and for SD dogs ranged from 0 to 3.
Immunofluorescent antibody titer and STAT-PAK results in 46 dogs at high risk for T cruzi infection.
IFA titer | Total tested | STAT-PAK positive | STAT-PAK negative |
---|---|---|---|
0 | 25 | 8 | 17 |
20 | 0 | 0 | 0 |
40 | 3 | 2 | 1 |
80 | 3 | 3 | 0 |
160 | 0 | 0 | 0 |
320 | 5 | 4 | 1 |
640 | 4 | 4 | 0 |
> 1,280 | 6 | 6 | 0 |
Most dogs had cTnI concentrations below the reference range (40 of 46 [87%]; Table 1). When compared between groups, cTnI concentrations in the SP group were significantly higher than in the SD group (Figure 1). Two dogs (1 SP, 1 SD) had cTnI concentrations > 2 times the upper limit of the reference range; the SD dog was also PCR positive while the SP dog was not. In the SP group, 1 of the 4 dogs with elevated cTnI was PCR positive. One dog in the SN group with cTnI below the reference range was PCR positive, suggesting a possible recent infection prior to developing a serologic response. This dog did not have any ECG or echocardiographic abnormalities.
Cardiac diagnostics
The CardioPet computer-aided program classified the ECG as abnormal in 29 of 46 (63%) and detected arrhythmias in 2 of 46 (4%). Upon investigator review, 19 of 46 (41%) were classified as abnormal and an arrhythmia was detected in the same 2 dogs. Five dogs had conflicting normal or abnormal classifications, with 4 that were normal on investigator review called abnormal by the machine and 1 called abnormal by the investigators for prolonged P wave duration in an SD dog. In 24 dogs, manual adjustments were made to automated ECG measurements predominately related to QRS and QT interval durations. Rarely, manual adjustments were made for PR duration (n = 2), P wave amplitude (2), and R wave amplitude (2) in 3 dogs with splintered QRS complexes (2) or artifact on the ECG recording (1). The most common ECG abnormality was R wave amplitude exceeding 3 mV in 10 dogs identified across all 3 groups (Table 1; Figure 1). Only 4 of 10 dogs with a tall R wave had increased left ventricular measurements on echocardiography indicating enlargement. No other ECG abnormalities were documented in the SN dogs, including 1 that was PCR positive and 1 that had an elevated cTnI concentration. Additional ECG abnormalities were detected in 8 of 29 (28%) dogs from the SP and SD groups (7 SP, 1 SD) and included prolonged P duration in 4 dogs (3 SP, 1 SD), ventricular premature complexes in 2 SP dogs, splintered QRS complexes in 2 SP dogs (1 of which had an R wave amplitude < 0.5 mV; Figure 2), prolonged PR interval in 1 SP dog indicating first-degree AV block, and intermittent second-degree AV block in 1 SP dog. Four dogs, all SP, had > 1 ECG abnormality. No dogs had supraventricular premature complexes or third-degree AV block. Five of the 6 dogs with elevated serum cTnI concentration had ECG abnormalities. Three were SP with conduction abnormalities, and 2 (1 SP, 1 SD) had ventricular arrhythmias noted during the echocardiogram only.
Echocardiographic abnormalities were documented in 26 (57%) dogs across all 3 serologic groups (Table 1). Fourteen of 46 dogs (30%) had evidence of myxomatous mitral valve disease (MMVD), with an age range of 2 to 11 years (median, 7.5 years). Left ventricular enlargement based on LVIDdN above the 95% confidence limit was noted in both the SP (n = 2, 1 of which had MMVD) and SN (3, 1 of which had MMVD) groups (Figure 1). Additionally, left ventricular systolic dysfunction based on at least 1 parameter (LVIDsN or E-point septal separation above the reference range or FS below the cutoff) was noted in 11 dogs in both the SP (n = 4) and SN (7) groups. Four dogs (1 SP, 3 SN) had both LVIDdN and LVIDsN above the 95% confidence limit. The single dog with FS below the cutoff also had an increased LVIDsN and was SN and PCR negative. Two dogs with left ventricular enlargement and left ventricular systolic dysfunction had an elevated cTnI concentration (1 SP, 1 SD). Neither left nor right atrial enlargement was detected in any dog. One SN dog had right ventricular enlargement based on mildly increased right ventricular internal diameter in diastole indexed to body weight. Four dogs (3 SP, 1 SD) had single ventricular premature complexes noted during the echocardiogram, 2 of which also had ventricular arrhythmias recorded on the ECG.
In exploration of the relationship between seropositivity (considering SD as SN) and cardiac abnormalities by lasso, only conduction abnormality was retained in the optimal multivariable model. In contrast, if SD cases were considered as SP, conduction abnormality, ventricular arrhythmias, increased cTnI concentration, increased LVIDdN, and increased LVIDsN were all retained in the optimized model. These relationships were difficult to evaluate further because they were exclusively associated with SP status. No ECG or echocardiographic abnormalities were detected in 12 of 46 (26%) dogs (5 SP, 4 SN, 3 SD).
Discussion
Our study documented ECG abnormalities more often in the SP and SD dogs than in the SN dogs in this population of dogs at high risk of T cruzi infection. While tall R wave was the most common ECG abnormality detected, it was present across all 3 serostatus groups, and despite its capability to increase with left ventricular enlargement,36 it was present in dogs with and without left ventricular enlargement in this study. Therefore, it did not distinguish between serostatus. When tall R wave was excluded, the remaining ECG abnormalities were detected exclusively in SP and SD dogs. These ECG abnormalities included conduction system delays (prolongation of the P, PR, and QRS interval durations; second-degree AV block; splintered QRS), decreased R wave amplitude, and ventricular arrhythmias. Conduction system abnormalities and ventricular arrhythmias are some of the earliest and most consistent changes observed throughout all stages of Chagas disease in humans, mice, macaques, and dogs.2,8,37,38 The range of ECG abnormalities is attributed to the widespread damage that can occur in any area of the heart due to the parasitic infection and development of myocarditis and myocardial fibrosis.1,2 Prolonged interval durations are frequently described in studies of infected dogs. In experimentally infected dogs, prolonged P and PR intervals were associated with histopathologic evidence of myocardial scarring, suggesting that what can appear to be minor ECG changes can be indicative of more severe, chronic myocardial damage.5 The presence of AV block was significantly associated with reduced survival time in 33% (14 of 42) of SP dogs presenting to a veterinary medical teaching hospital in Texas.8 In the same study, AV block and prolonged QRS interval duration (≥ 0.07 seconds) were associated with cardiac-related death.8 None of the dogs in our study with prolonged P wave duration (3 SP, 1 SD) had atrial enlargement. This highlights that while changes to the ECG complex can be attributed to chamber enlargement, they can also be indicators of a disease process that leads to prolonged interval durations and abnormal complex morphology with normal heart size.39
Ventricular arrhythmias are one of the most common abnormalities identified in dogs with Chagas disease.3,8,13 In dogs that presented to a veterinary medical teaching hospital in Texas, the odds of being infected with T cruzi were 2 times greater when ventricular arrhythmias were present when compared to arrhythmias originating from other locations and 3 times greater when dogs had arrhythmias arising from > 1 location including the ventricles.12 More complex ventricular arrhythmias with a higher modified Lown score (≥ 2) were significantly associated with reduced survival time and cardiac death in infected dogs.8 Ventricular arrhythmias were detected in 8% (4 of 46) of dogs in our study, all of which occurred as single ventricular premature complexes during the 30-second recordings or the echocardiogram. Ambulatory ECG (Holter) can provide more comprehensive information about the frequency and severity of ventricular arrhythmias compared to a standard ECG due to the longer recording time.8 However, conduction system abnormalities including prolonged durations and abnormal ECG complex morphology are more readily detected with a standard ECG recording.
The ECG performed in this study utilized the CardioPet system, a computer-assisted software for ECG analysis, to obtain 30-second ECG tracings for detection of arrhythmias and conduction abnormalities. The CardioPet device is available for use by veterinary practitioners and includes an option for virtual interpretation by a veterinary cardiologist. It not only facilitates acquisition of the ECG and measurements but also automatically classifies the tracing as normal or abnormal on the basis of computer-generated measurements and identification of arrhythmias. However, despite the advantages of automated analysis, oversite with the review of automated measurements is necessary for manual correction of misplacement of markers when needed. This is especially important when assessing conduction abnormalities, which require detailed assessment of the ECG including detection of prolonged interval durations and abnormal complex morphology. In our study, 5 dogs were classified as abnormal by the CardioPet but were considered normal on investigator review. In 2 dogs, the CardioPet accurately detected arrhythmias, consistent with previous findings indicating that the CardioPet system detects arrhythmias more reliably than abnormal ECG complex measurements when compared to cardiologists’ assessments.40
Echocardiographic abnormalities were documented in more than half of the dogs and were not restricted to dogs in the SP group. The SP group was significantly older than the SD and SN groups and was the group with the highest number of dogs with MMVD, which is an age-related degeneration of the valves. The presence of MMVD can result in left-sided heart enlargement, as can T cruzi infection, making it challenging to distinguish the underlying source of enlargement in some dogs. Mild left ventricular enlargement and ventricular systolic dysfunction were present in a small number of dogs without MMVD in this study. While causes of a dilated cardiomyopathy phenotype include myocarditis, there are other potential underlying etiologies to consider, including genetic mutation, diet associated, tachycardia induced, and various toxins and medications. Myocardial fibrosis associated with chronic myocarditis can manifest echocardiographically as wall motion abnormalities, global systolic dysfunction, or changes in the appearance of the myocardium (ie, abnormal echogenicity); however, these can be more readily detected with cardiac MRI.18 Only 1 dog had both increased LVIDsN and reduced FS and was in the SN group with negative PCR results. There was not any evidence of atrial enlargement in this group of asymptomatic dogs. Right ventricular enlargement in Chagas disease is associated with a higher likelihood of having clinical signs and is an indicator of advanced disease with decreased survival times reported in dogs.2,8,15 The only dog in this study with mild right ventricular enlargement was in the SN group, possibly indicating a false-negative serologic test result or enlargement that occurred from disease unrelated to T cruzi infection.
Cardiac troponin I is released from damaged cardiomyocytes, and elevations are considered both a sensitive and specific indicator of myocardial damage.41 Thus, cTnI can serve as a biomarker of myocardial damage, although elevations are not specific to T cruzi alone. In our study, 13% (6 of 46) of dogs across all 3 serostatus groups had an elevated cTnI concentration. An elevated cTnI concentration in a SN dog could occur with an acute T cruzi infection prior to seroconversion; however, other causes of elevated cTnI are possible and include many other cardiac causes, infectious causes, immune-mediated disease, systemic disease, toxins, and trauma.41 Increased cTnI concentrations have been documented in dogs infected with T cruzi, although the degree of elevation can vary with stage and severity of disease.8,13,15,16,18 Most dogs in our study had cTnI concentrations below the reference range, which is consistent with the low FETCH scores and absence of reported clinical signs. In our experience, elevated cTnI concentrations are more likely to be detected in acute or active T cruzi infections.15,16 Dogs with elevated cTnI concentration from any cause would likely benefit from further evaluation or more frequent monitoring.
Twenty-two percent of dogs were classified as SD, highlighting the challenge of making a diagnosis when test results do not agree. When dogs in the SD group were included with the SP group, SP status increased to 63%. In humans, the use of confirmatory testing improves the confidence of diagnosis and is recommended when testing for Chagas disease to reduce discordant test results, which can occur due to parasite heterogeneity and host factors.19,42 The IFA test utilized in this study is available in a reference laboratory and has been validated in dogs, while the STAT-PAK is a rapid test utilized in dogs that has been validated using human sera from Central America in which circulating T cruzi strains may differ from those in Texas.21 While point-of-care tests have the potential benefit of making a more rapid diagnosis as a screening tool, they can also have challenges with interpretation of faint bands and are susceptible to false-positive results.13,43 As additional tests for T cruzi become available for dogs, comparison of test results to confirm infection will be important, as there is no gold-standard test or diagnostic algorithm for Chagas disease in dogs. Further work is needed to validate diagnostic assays and standardize the diagnostic assessment of dogs with T cruzi infection to help clarify infection status.44 Most dogs in our study were considered chronically infected on the basis of SP status, predominately negative PCR, low serum cTnI concentrations, and absence of clinical signs. In humans, the chronic indeterminate stage was defined in 1985 as SP status, absence of clinical signs, and normal ECG; however, ECG abnormalities can be intermittent and discovered with routine monitoring.45,46 No abnormalities were detected with ECG or echocardiogram in 5 SP dogs in this study. In humans, this is considered to convey a more favorable prognosis, with routine monitoring still recommended.47 Additionally, SP dogs were significantly older than the dogs in the SN or SD group, a finding that is documented in dogs and humans in areas with infected kissing bugs related to increased risk of exposure over time.48–50 Results from PCR can detect parasite DNA in the blood of infected dogs; however, parasitemia declines after the acute phase of disease, producing disagreement between serologic and PCR tests.2,11 In this study, 19% (9 of 46) of dogs were PCR positive, with 1 PCR-positive dog in each of the SD and SN groups. Positive PCR results were considered evidence of acute infection or active parasitemia. Acute infection was considered possible in the SN, PCR-positive dog.
This study had several limitations. Dogs enrolled in the study did not have clinical signs on the basis of owner-provided FETCH scores. However, dogs in these types of working environments are rehomed or culled from the population if they have clinical signs and are not able to do their job. This could have influenced which dogs were available to be enrolled and thus led to sampling bias affecting study results. Age was estimated for some dogs without a known date of birth. Additionally, results were based on serostatus; however, positive blood PCR results were documented in dogs in both the SD and SN groups. This may have indicated acute infection or false-positive PCR results but was not able to be followed up with repeat serology testing to reassess serostatus. The ECG recording in this study was limited to 30 seconds and could have missed intermittent arrhythmias that would have been detected with a 24-hour ambulatory ECG recording. While a more comprehensive diagnostic evaluation may have documented additional cardiac abnormalities, the goal of this study was to evaluate a simplified diagnostic evaluation. It is also important to note that ECG and echocardiographic abnormalities and cTnI elevations are not specific to Chagas disease and can occur for many other reasons in dogs that were not explored in this study. Additionally, tests for other vector-borne or infectious diseases were not performed at the time of this study to rule other potential causes of myocarditis or cardiac diagnostic test abnormalities. Dogs from our study were residing in the same area at the time of the study. Considering the diverse T cruzi strains observed across different regions with varying degrees of pathogenicity, the findings from our study might not apply to populations in other geographic areas.
In summary, the presence of conduction system abnormalities and ventricular arrhythmias may be more common in seroreactive dogs, prompting further diagnostic evaluation and monitoring for the development of Chagas disease. The detection of conduction system abnormalities requires an assessment and detailed measurement of the ECG complexes. While there are benefits to using an automated system like the CardioPet device to acquire the ECG, oversite with review of automated measurements is necessary to correct misplacement of markers in dogs with altered complex morphology. Further study would be required to determine whether the results of this study could be applied to a larger population of dogs.
Acknowledgments
The authors would like to thank Cyneen Santoya, Kristen Flitcroft, Cerina Ferguson, Rachel Busselman, and Angela Molli for assistance with data collection and sample processing and Idexx Laboratories for providing the CardioPet ECG machine.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
Funding for the study was provided by the George A. Robinson Foundation and the DAKOTA Fund.
References
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