Cardiac biomarkers in dogs with visceral leishmaniasis
Biomarcadores cardíacos en perros con leishmaniasis visceral
a Federal University of Paraná, Department of Veterinary Medicine, Paraná, Brazil.
ABSTRACT. Although only a few studies have focused on the cardiac alterations in Leishmania spp. infected dogs, myocardial inflammation ascribed to such parasite has been documented in some animals. In spite of the increasing popularity of cardiac biomarkers in veterinary medicine in recent years, it remains unknown how most of these substances would perform in dogs with visceral leishmaniasis. In this study, we hypothesised that Leishmania spp. would damage cardiac myofibers resulting in increased circulating levels of cardiac specific markers in dogs. Therefore, biomarkers of myocyte injury and stress were investigated in 18 dogs with naturally-occurring visceral leishmaniasis presenting varying clinical signs, including completely asymptomatic animals. The median (min-max) concentrations of NT-proANP, NT-proBNP, cTnI, and CK-MB were 1138.0 (875-1175) pmol/L, 1160.0 (803-2034) fmol/L, 0.22 (0.15-0.51) ng/mL, and 116.7 (113-222) U/L, respectively. No differences were documented for the concentration of the natriuretic peptides and CK-MB in accordance with the severity of clinical signs, but for cTnI a significant difference was reported when comparing asymptomatic and symptomatic animals. Also, the compromise of renal tubular function seemed to interfere with the concentration of the natriuretic peptides and cTnI. In conclusion, all cardiac-specific biomarkers were elevated when compared to the normal range previously proposed for dogs. Measuring specific cardiac biomarkers has the potential to non-invasively document myocardial injuries attributable to visceral leishmaniasis.
Key words: parasitic disease, myocarditis, natriuretic peptides, troponins.
RESUMEN. A pesar de que solo unos pocos estudios se han centrado en las alteraciones cardíacas en perros infectados por la Leishmania spp., la inflamación miocárdica atribuida a este parásito ha sido documentada en algunos animales. Los marcadores cardiacos en medicina veterinaria han experimentado una creciente popularidad en los últimos años, sin embargo aún no se sabe cómo la mayoría de estas substancias se comporta en perros con leishmaniasis visceral. En este estudio, se presume que Leishmania spp. dañaría fibras cardiacas causando un aumento de los niveles circulantes de marcadores cardiacos en perros. Biomarcadores de lesión y de estrés de los miocitos fueron investigados en 18 perros con infección natural por Leishmania spp. y que presentaban diversos signos clínicos, incluyendo los animales totalmente asintomáticos. La mediana (mín-máx) de las concentraciones de NT-proANP, NT-proBNP, cTnI, CK-MB fueron 1138,0 (875-1175) pmol/L, 1160,0 (803-2034) fmol/L, 0,22 (0,15 -0,51) ng/mL y 116,7 (113-222) U/L, respectivamente. No se registraron diferencias para la concentración de los péptidos natriuréticos y CK-MB de conformidad con la gravedad de los síntomas clínicos, pero para la cTnI se observó diferencia significativa al comparar los animales sintomáticos y asintomáticos. Además, el compromiso de función tubular renal parece interferir con la concentración de los péptidos natriuréticos y la cTnI. En conclusión, todos los cardiobiomarcadores específicos estaban elevados en comparación con el rango normal anteriormente propuesto para perros. La cuantificación de determinados biomarcadores cardíacos tiene el potencial de identificar, de forma no invasiva, lesiones miocárdicas atribuidas a la leishmaniasis visceral.
Palabras clave: enfermedad parasitaria, miocarditis, péptidos natriuréticos, troponinas.
Visceral leishmaniasis is a potentially fatal zoonotic disease that occurs in people and both domestic and wild animals. It is caused by the protozoa Leishmania (Leishmania) infantun chagasi, which is disseminated through phlebotomine sand flies. Although not frequently mentioned in literature, lesions in the cardiovascular system have been documented in Leishmania infections, and therefore should be considered as part of the several alterations that might be caused by such parasite. Both dogs and human beings have been reported to develop a myocarditis attributable to Leishmania spp., which is histologically characterised by edema, inflammatory infiltration, focal necrosis, and Anitschkow myocytes proliferation (Torrent et al 2005, Baneth et al 2006, Lopez-Pena et al 2009, Alves et al 2010).
When the necrotic myocytes lose their integrity, macromolecules leak into the interstitial tissue, following absorption by the capillary bed and lymphatic system. Such molecules are considered biological markers or biomarkers, which are defined as substances that may be measured objectively as a surrogate for either normal or pathological processes, or even change their concentration in response to therapeutic interventions. Indeed it is desired that biomarkers could be produced by a specific tissue, being easily detected when released into the circulation (Lopez-Sendón 2003, Reynolds and Oyama 2008).
In a prior study we investigated cardiac rhythm disturbances and ECG wave abnormalities in 105 dogs with confirmed visceral leishmansiasis and found sinus arrest, right bundle branch block, and atrial premature beats in 14.3%, 4.8%, and 4.8% of the studied subjects, respectively (Sousa et al 2013). Also, when hearts of 30 dogs with the same disease were assessed histologically and immunohistochemically, Rosa et al (2013) found lymphoplasmacytic myocarditis, myonecrosis, and increased interstitial collagen in the majority of the animals, and Leishmania spp. could be documented in 66% of the animals, which likely points to myocardial lesions and their consequences being attributable to such parasite.
To the authors' knowledge, cardiac specific biomarkers have not been fully investigated in dogs with naturally-occurring visceral leishmaniasis, although they do have been studied in other parasitic and infectious diseases. A study by Barr et al (2009) investigated the serum concentration of cardiac troponin I in a model of acute myocarditis in dogs infected with Trypanosoma cruzi, a protozoa belonging to the same family of Leishmania spp.. In that study, an association was found to exist between cTnl levels and the myocardial injury seem at the histopathological assessment of the dog's hearts. Therefore, it is reasonable to hypothesize that alterations in cardiac myofibers could be documented by the circulating levels of cardiac specific markers in dogs naturally-infected by Leishmania spp.. Also, we sought to determine whether the clinical status of the animals could potentially interfere with the concentration of these substances.
MATERIAL AND METHODS
ANIMALS AND GROUPS
Eighteen mature mixed-breed dogs (2.7-32.1 kg; 9 males) diagnosed with visceral leishmaniasis were prospectively included in a cross-sectional observational study. The diagnosis of leishmaniasis was based on the demonstration of amastigotes of Leishmania spp. in lymph node aspirates, as well as a positive serology in both ELISA and IIFA. Exclusion criteria included the animal testing positive in serology for leptospirosis, toxoplasmosis, neosporosis, ehrlichiosis, babesiosis, and heartworm, Lyme and Chagas diseases, as well as the owner not voluntarily agreeing to have the animal enrolled in the investigation, which was entirely conducted within a Veterinary Teaching facility. Once the dogs were recruited into the study, clinical signs were recorded to allow their categorization in asymptomatic, olygosymptomatic (a maximum of two clinical signs), or polysymptomatic (more than two clinical signs) animals. Varying clinical signs were observed, including emaciation, lymphadenomegaly, skin lesions, ocular alterations, muscle atrophy, and onicogryphosis, but some of them had no clinical manifestations at all.
The study was entirely conducted in accordance with the guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the institutional animal experimentation ethical committee (10/1621).
QUANTIFICATION OF CARDIAC BIOMARKERS
Blood samples were drawn to obtain both the plasma and serum. Several aliquots were stored at -80oC until the batch analyses were performed. The plasma concentrations of N-terminal pro-ANP (NT-proANP), N-terminal pro-BNP (NT-proBNP), and MB fraction of creatine kinase (CK-MB), as well as the serum concentration of cardiac troponin I (cTnI) were measured using commercially available kits (CSB-EQ027254DO, Cusabio, Wuhan, China; E90485CA, Uscn Life Sciences Technology, Missouri City, EUA; CSB-E08772C, Cusabio, Wuhan, China; 118, Labtest, Lagoa Santa, Brazil). The kits used for measurement of natriuretic peptides and cardiac troponin I were all species specific. All manufacturer recommendations were strictly followed for laboratory procedures involved with the measurements.
OTHER BIOCHEMICHAL ANALYSES
Blood and urine samples were used for quantification of creatinine concentration, urine protein-to-creatinine ratio (UPC), and the fraction excretion (FE) of sodium and potassium. These analyses were undertaken using standard laboratory methods immediately after the animals were admitted into hospital. UPC was documented as the ratio between urine protein and urine creatinine, and the FE of electrolytes was determined using the following equation: FE = [(Urine electrolyte • Plasma creatinine) / (Urine creatinine ∙ Plasma electrolyte)] ∙ 100.
Descriptive statistics were used to describe population characteristics. The results underwent the Shapiro-Wilk normality test, followed by either an analysis of variance for parametric data or the Kruskal-Wallis test for non-parametric data to compare animals in accordance with their presenting clinical signs. Either Pearson or Spearman test was used to look for individual correlations between the blood concentration of the several biomarkers and body weight, serum creatinine concentration, urine protein-to-creatinine ratio, and the fraction excretion of sodium and potassium. Statistical analyses were performed using statistical software Graphpad Prism, v.5.0 (California, USA), and significance was set at P < 0.05 for all analyses.
Table 1 shows the descriptive statistics of the blood concentration of the several cardiac biomarkers that were studied. Elevated concentrations of NT-proBNP (> 800 pmol/L), cTnI (> 0.03 ng/mL), and CK-MB (> 6.3 U/L) were documented in all animals enrolled in the study, but increased NT-proANP (> 1,000 pmol/L) was found in only 66.6% (12/18) of the dogs. However, when all subjects were considered together, the mean values of all four cardiac specific biomarkers were above the reference range for normal dogs (Montes et al 1987, Sleeper et al 2001, Ettinger and Prosek 2010, Eriksson et al 2014).
Table 1. Descriptive statistics showing the blood concentration of cardiac biomarkers in
CI: confidence interval.
Although no differences were documented in the concentration of NT-proANP, NT-proBNP, and CK-MB according to the group to which the animals were assigned, there is an overt trend of increase in such concentrations as the clinical scenario worsened (table 2). For cTnI, a significant difference was attained when comparing asymptomatic and symptomatic animals (figure 1).
Table 2. Cardiac biomarker characteristics in dogs with visceral leishmaniasis grouped
Different letters in the same line indicate significant differences at the post hoc test.
Figure 1. Individual values and medians of the blood
The results of blood and urine biochemical analyses are shown on table 3. Although no correlation existed between cardiac biomarkers and body weight, weak to moderate correlations were found to exist between the Na+ fraction excretion and the blood concentration of NT-proBNP and cTnI, as well as between the K+ fraction excretion and the concentrations of NT-proANP, NT-proBNP, and cTnI (figure 2).
SD: standard deviation; CV: coefficient of variation; FE: fraction excretion.
Figure 2. Scatter plots of the blood concentration of NT-proANP (A), NT-proBNP (B, C),
Several infectious and noninfectious diseases might result in compromise of the heart muscle, including parvovirus infection, heartworm disease, endocarditis, and dilated cardiomyopathy (Sharkey et al 1991, Santos 2005, Diniz et al 2007). Despite not being frequently documented, some studies have shown that Leishmania (Leishmania) infantum chagasi infected dogs may develop some sort of myocardial injury (Torrent et al 2005, Lopéz-Peña et al 2009, Alves et al 2010, Sousa et al 2013). Because studies have found contrasting results regarding the identification of Leishmania spp within the myocardium of these animals, it might be speculated that the basis for myocardial injury in infected subjects involves the inflammation ascribed to direct muscular injury from the parasite, as well as immune-mediated myositis (Gomes et al 2012, Rosa et al 2014)
Natriuretic peptides are biomarkers of cardiac remodeling and congestive heart failure, which have been shown, in recent years, to aid in establishing both the diagnosis and prognosis of several cardiac diseases (Maack 2006, Richards 2007). The N-terminal proANP was once demonstrated to have a greater reliability in assessing conditions such as chronic mitral regurgitation, the cardiac remodeling attributable to volume overload, heartworm disease, and atrial fibrillation (Shober 2005, Ettinger and Prosek 2010). Also, Prosek et al (2007) documented a sensitivity of 95.5% and specificity of 84.6% for the NT-proANP in differentiating dogs with dyspnea ascribed to either cardiac or non-cardiac conditions.
Several studies have evaluated the relationship between the blood concentration of NT-proBNP and the degree of cardiac compromise in many heart problems (DeFrancesco et al 2007, Serres et al 2009, Moonarmart et al 2010). It was shown that the more severe the heart failure, the higher the circulating levels of this peptide. Asymptomatic mitral valve dogs bearing increased levels of NT-proBNP developed signs of congestive heart failure more rapidly than the asymptomatic patients that had a lower blood concentration of that biomarker. Also, it was demonstrated that the augmented levels might indicate an increased probability of cardiac-related death.
NT-proBNP values ranging from 800-1800 pmol/L are highly suggestive of cardiac disease (Ettinger and Prosek 2010). Despite no significant differences in NT-proBNP concentration had been documented between the groups of this study, all animals tested within the range which might indicate cardiac involvement. Interestingly, there is a trend of increase in the blood concentration of such biomarker as the animals' clinical scenario worsened. A similar finding was reported by Lobetti et al (2012), who investigated the serum concentrations of NT-proBNP and cTnI in 45 dogs with babesiosis. In that study, the concentration of the biomarkers increased along with the severity of the disease.
To the authors' knowledge, no other study investigated how NT-proANP performs in dogs with either infectious or parasitic diseases. However, several studies have proven its reliability in detecting cardiac failure in dogs with heart disease (Ettinger and Prosek 2010, Eriksson et al 2014). In this investigation, NT-proANP was the only cardiac specific biomarker that was not elevated in all recruited dogs. Nonetheless, its concentration was found to be above the reference range (> 1.000 pmol/L) in 66.6% of the animals, being consistent with the increased concentrations of the other biomarkers.
Cardiac troponin I, which is found freely in the cytosol and accounts for approximately 2 to 4% of the total myocardial troponins, has been recognised as a highly sensitive and specific biomarker for myocardial injury in human beings (Apple et al 1998) as well as in animals (Schober et al 1999). In a study that included 42 critically ill dogs without primary cardiac compromise, cTnI was able to predict the short term death (Langhorn et al 2013).
Immunoassays have been validated to determine the blood concentration of cTnI in dogs, which indicated the normal range in that species to be < 0.03 ng/mL (Schober et al 1999, Sleeper et al 2001). The concentration of cTnI for all animals recruited for this study was above the reference range for normal dogs. Although no differences existed between asymptomatic and olygosymptomatic dogs, an increasing concentration of cTnI was seen in accordance with the decline in the clinical status of the dogs. Thus, the polysymptomatic animals presented a concentration that far exceeded the normality range, suggesting intense myocyte damage. In a similar study, Silvestrini et al (2012) has already documented an increased concentration of blood cTnI when comparing dogs with visceral leishmaniasis and control animals. Also, 40% of the infected animals in their investigation had the cTnI concentration above the normal reference range, which pointed to some degree of myocardial injury. Recently, Mendes et al (2014) reported a case of a dog with naturally-occurring visceral leishmaniasis, in which the cTnI concentration (0.22 ng/ mL) was identical to the mean value documented for the olygosymptomatic group of this investigation.
Other infectious diseases are known to result in increased levels of cTnI as well. Güneş et al (2014) found elevated cTnI and cTnT in dogs with parvovirus infection and distemper, which reinforces how valuable the measurement of cardiac troponins may be when myocardial injury is suspected. When dogs with heartworm disease were studied, Carretón et al (2014) showed a progressive increase in cTnI in accordance with the severity of the disease. Also, dogs with naturally occurring ehrlichiosis were shown to have elevated cTnI when compared to healthy controls (Diniz et al, 2008).
Cardiac troponins have demonstrated high sensitivity in diagnosing noninfectious conditions as well. Shaw and colleagues (2004) demonstrated its utility in differentiating idiopathic pericardial effusion and the pericardial effusion ascribed to hemangiossarcoma, with the latter presenting increased values of cTnI as compared to the former. Using a combination of clinical information, cTnI concentration and electrocardiographic findings, Diniz et al (2007) diagnosed two cases of myocarditis in dogs that experienced chest trauma.
Creatine kinase MB isoenzyme is released into the extracellular space by necrotic myocardial cells, which potentially allows its use as a surrogate for cardiac injury (Schober et al 1999, Diniz et al 2007). Dogs with cardiovascular diseases have been shown to present significantly higher levels of CK-MB in comparison with healthy subjects, being this condition attributable to alterations in membrane permeability owing to myocyte instability and hypoxia (Pino et al 2008). Both Schober et al (1999) and Diniz et al (2007) reported augmented CK-MB concentrations in dogs suspected of blunt cardiac injury. However, Diniz et al (2007) mentioned that CK-MB might have been released from either the myocardium or the skeletal muscle, since the animals reported in their study had concurrent muscle injury.
In dogs with visceral leishmaniasis, Santos (2013) documented an increased mean concentration of CK-MB (450.9 U/L), which was much higher than both their healthy controls and the infected dogs of this investigation. Also contrasting with the results of this study, a recent investigation in which 41 dogs with visceral leishmaniasis had been categorised into asymptomatic, olygosymptomatic, and symptomatic groups, reported greater concentrations of CK-MB in all groups (263, 488.4, and 681.2 U/L, respectively) (Godoy 2015). However, every animal of this investigation had a CK-MB concentration that far exceeded the reference range of 4.9 to 6.3 U/L previously proposed for normal dogs (Montes et al 1987).
In this study, we also investigated the correlation of several parameters used to assess both glomerular and tubular functions and the blood concentration of the cardiac biomarkers, since the altered renal function ascribed to Leishmania spp. (Costa et al 2003, Baneth et al 2008) may interfere with the concentration and clearance of those substances. The electrolyte fraction excretion, which is defined as the relationship between any given electrolyte and the creatinine elimination, may be used as a surrogate for renal tubular function (DiBartola 2000, Martinez and Carvalho 2010). Thus, the significant positive correlations between the Na+ and K+ fraction excretions and NT-proANP, NT-proBNP, and cTnI might be explained, at least in part, by the deterioration of renal function (Freda et al 2002, Raffan et al 2009) and not to a true myocardial injury. However, since no histopathological assessment of the hearts was performed in this study, we could not investigate whether myocardial inflammation and structural damage were present at all.
Among the several limitations of this study are the small number of dogs enrolled, which may have compromised the ability of the statistical analysis to document differences in the blood concentration of the several cardiac biomarkers between asymptomatic and symptomatic animals. Also, the absence of an echocardiographic assessment did not allow correlating any changes in myocardial function and structure with the biomarkers concentrations. Finally, since only a few infectious and parasitic comorbidities have been ruled out, our results could represent the effect of other systemic conditions over the heart.
In conclusion, this study documented elevated cTnI in all dogs, as well as increased NT-proBNP in animals with visceral leishmaniasis. The concentration of cardiac troponin I became significantly higher as the clinical scenario worsened. Although not significant, a trend of increase was also observed for the remainder of cardiac biomarkers. The quantification of specific cardiac biomarkers is likely useful to identify myocardial injury attributable to visceral leishmaniasis.
The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the financial support of this study (grant number 309523/2012-5).
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