Canine Visceral Leishmaniasis in an Area of Sporadic Transmission in Brazil

Abstract

Canine visceral leishmaniasis is a zoonotic disease caused by the protozoan Leishmania infantum in Latin America. Visceral leishmaniasis (VL) diagnosis in Brazil includes two serological tests according to the Ministry of Health (MH) protocol. Sensitivity and specificity of diagnostic tests, as well as clinical signs of VL, are usually reported in disease-endemic areas; however, it is known that local epidemiological factors can influence these results. This study aimed to evaluate the clinical features, sensitivity, and specificity of TR-DPP^® and EIE-LVC in naturally infected dogs in a region of sporadic VL transmission to humans in Brazil. A total of 288 dogs were clinically evaluated and serological and parasitological (lymph node aspirates) samples were collected for VL diagnosis. TR-DPP and EIE-LVC showed poor sensitivity (0.62 and 0.44, respectively) to detect infected animals, compared with the direct parasitological examination, which is considered a gold standard method. Thus, the protocol of MH presented low sensitivity (0.42) to estimate prevalence and control measures in this region. TR-DPP presented a high negative predictive value (0.89), resulting in its indication as a confirmatory test in sporadic transmission areas. Classical clinical signs of VL were not frequently observed; therefore, clinical scoring systems might not be useful in this region. Veterinarians of nonendemic areas should be alert for asymptomatic dogs, especially those presenting lymph adenomegaly.

Introduction

Visceral leishmaniasis (VL) is caused by several species of the Leishmania donovani complex, which is transmitted during bloodmeals of female sand flies (Diptera: Psychodidae: Phlebotominae) from the genera Phlebotomus (Old World: Asia, Africa, and Europe) and Lutzomyia (New World: Americas) (Killick-Kendrick 1990, Cox 2005, Pan American Health Organization (PAHO) 2019). In South America, the disease is considered an anthropozoonotic disease and its etiologic agent is similar (Fernández-Arévalo et al. 2020) to Leishmania infantum (Nicolle 1908), the species responsible for zoonotic/anthroponotic VL in the Mediterranean area (Gradoni 2017). In Brazil, L. infantum [senior syn. Leishmania chagasi (Cunha and Chagas 1937)] is the etiological agent of VL and the sand fly, Lutzomyia longipalpis (Burza et al. 2018), is the main vector. In some particular Brazilian areas, Lutzomyia cruzi has been accepted as the vector of L. infantum (Oliveira et al. 2017a,b).

VL is considered one of the most severe parasitic diseases in the world, considering its burden and potential to reduce the productive years of an individual, leading to death (Kassebaum et al. 2016, Ruiz-Postigo et al. 2020). In 2018, the endemicity status for the disease was reported in 78 countries and territories, according to WHO data (Ruiz-Postigo et al. 2020), and an estimated 30,000 new cases occur annually (PAHO 2019). In the last few decades, an increase in mortality and lethality due to introduction of the disease in new geographic areas and unfavorable host factors, such as malnutrition, immunosuppression, and other comorbidities, was observed (Martins-Melo et al. 2018). In Brazil, VL relevance relies on the disease's heterogeneous pattern of transmission and the country's participation in almost 99% of the cases registered in Latin America (Burza et al. 2018). Besides, the disease has a significant economic impact on public health expenditure, accounting for approximately US$ 500.91/patient of the unified health system (Carvalho et al. 2017).

Among the control measures adopted to reduce VL spread in Brazil are vector control, early diagnosis and treatment for humans, and culling of seropositive dogs (Brazilian Ministry of Health 2014). Nowadays, the Brazilian Ministry of Health recommends an association of serological tests that search for IgG antibodies in domestic canines. The immunochromatographic rapid test (TR-DPP^®; Bio-Manguinhos/Fiocruz/Brazil) is used as a screening diagnostic tool, and confirmation is provided by the enzyme-linked immunosorbent assay (EIE-LVC) (Brazilian Ministry of Health 2011, 2017). However, the disease prevalence in a specific area can interfere with sensitivity and specificity of serological tests for canine visceral leishmaniasis (CVL) diagnosis. This fact represents additional challenges for disease recognition in low prevalence regions (Mendonça et al. 2017).

Domestic canines are considered important reservoirs of L. infantum due to the high parasitic load on their skin, which promotes vector infection despite their clinical presentation (Laurenti et al. 2013). Considering the variety of clinical manifestations presented by infected animals, accurate diagnosis of CVL has become one of the major obstacles in its control. Many clinical classifications and score systems have been developed in endemic regions to support CVL diagnosis (Mancianti et al. 1988, Solano-Gallego et al. 2009, Silva et al. 2017). However, the clinical signs presented in CVL are also compatible with other pathologies, making clinical diagnosis of CVL unfeasible (Pinelli et al. 1994, Quinnell et al. 2001, Moreno and Alvar 2002) and reinforcing the need for laboratory diagnosis.

In this sense, the lack of laboratory resources in some small Brazilian municipalities may postpone canine CVL diagnosis, and clinical signs might be the only starter tool toward diagnosis.

Considering the importance of early CVL diagnosis due to its potential as a marker of L. infantum transmission to humans (Paranhos-Silva et al. 1996, Magalhães-Junior et al. 2016), especially in regions of recent parasite introduction and low prevalence of human disease, this study aimed to evaluate the prevalence and clinical manifestations, as well as clinical features and diagnostic test performance, of CVL in an area of sporadic VL transmission in Brazil.

Materials and Methods

Ethical aspects

This project was approved by the Ethics Committee on Animal Use (CEUA) of the Federal University of Mato Grosso do Sul (UFMS) under certified protocol number 645/2014. Dogs' guardians included in this study agreed to sign an informed consent form.

Study area and population

The study was conducted from March 2015 to March 2017 in the municipality of Camapuã, Mato Grosso do Sul, Brazil (19° 31′ 38″ S; 54° 2′ 32″ W). According to the MH system for VL transmission status, Camapuã is considered as an area of sporadic VL transmission (mean of human cases over the last 5 years less than 2.4) (Mato Grosso do Sul 2017) and the vector involved in L. infantum transmission in this region is Lu. cruzi (Fernandes et al. 2017).

The sample comprised 288 dogs chosen by stratified sampling using conglomerates, as suggested by MH (Brazilian Ministry of Health 2014), which were obtained during a canine survey census conducted by the Municipal Health Department of Camapuã in July 2015. Dogs aged less than 4 months were excluded from the study due to maternal antibody interference.

Clinical assessment

All dogs were clinically assessed and classified according to the score protocol proposed by Silva et al. (2017). The presence or absence of a set of 14 different clinical signs (nutritional status; bristles; onychogryphosis; lymph node enlargement; mucosal color; snout lesions; snout depigmentation; muzzle lesions; muzzle depigmentation; ear lesion; skin lesion; keratoconjunctivitis; alopecia; and blepharitis) was used to classify the dogs. Based on the total score, each animal was classified as asymptomatic (zero to three points) or symptomatic (more than three points). During clinical evaluation of dogs, data such as age, gender (male or female), and hair features (short or long) were registered.

Collection of biological samples

From all animals, a 10-mL peripheral blood sample was collected by jugular venipuncture. Samples were stored in a tube containing silica clot activator to obtain serum. Lymph node aspiration puncture was performed with a 0.70 × 25-mm needle coupled to 3-mL syringe at popliteal lymph nodes. All 288 dogs were submitted to three diagnostic tests: immunochromatographic rapid test (TR-DPP); direct parasitological test and enzyme-linked immunosorbent assay (EIE-LVC).

This study was part of the epidemiological survey performed by the Municipal Health Surveillance Service of Camapuã. Therefore, performing molecular analysis in all samples would be financially impracticable, besides the absence of qualified manpower to perform it.

Direct parasitological diagnosis

Lymph node aspirate smear slides were stained with Giemsa and underwent visualization using light-field microscopy with a 100 × objective for direct parasitological diagnosis. The choice of lymph node samples was due to their sensitivity of 40–50% for direct parasitological examination, while bone marrow presents sensitivity of 60–75% (Alvar et al. 2004). Spleen puncture provides a rich material that achieves 98% sensitivity for diagnosis (Barrouin-Melo et al. 2004); however, bone marrow and spleen punctures are not considered routine procedures and would be inapplicable in epidemiological surveys (Gontijo and Melo 2004).

Serological diagnosis of CVL

Immunochromatographic rapid test: TR-DPP

The immunochromatographic rapid test, TR-DPP (Dual-Path Platform immunochromatographic test—CVL, Bio-Manguinhos/FIOCRUZ—Rio de Janeiro, Brazil), was performed with each animal's serum sample, in accordance with the instructions provided by the manufacturer. Ten microliters of each serum sample and 2 drops of buffer solution were added to well no. 1 of the platform. After 5 min, 4 drops of buffer solution were added to well no. 2. Test reading was performed after 10 min, two lines being an indication of reagent result. The presence of only one line represents the internal control reaction and therefore indicates a nonreagent result for the sample tested.

Enzyme-linked immunosorbent assay: EIE-LVC

The EIE-LVC to detect IgG anti-Leishmania antibodies was performed at the Central Laboratory of Public Health of the State of Mato Grosso do Sul, LACEN/MS, in Campo Grande, MS, using a commercial kit (EIE-LVC; Bio-Manguinhos, Fiocruz, Rio de Janeiro, Brazil).

Serum samples were diluted (1:100) in the provided diluent, and 100 μL of each diluted sample was distributed in a 96-well EIE-LVC-sensitized plate available in the kit, which was incubated at 37°C for 30 min. The plate was then washed six times with the kit wash buffer (200 μL/well). The conjugate was diluted in the diluent solution according to the manufacturer's orders and distributed in each well (100 μL/well), followed by incubation at 37°C for 30 min. The washing procedure was repeated, as previously mentioned. The substrate (H₂O₂) was distributed in each well (100 μL/well) and incubated at room temperature for 30 min. The reaction was blocked by adding 50 μL of sulfuric acid (2 M) in each well. Readings were performed with a spectrophotometer using a 450-nm filter.

Statistical analyses

Sensitivity, specificity, positive predictive value, and negative predictive value calculations were performed for TR-DPP and EIE-LVC^®, using the direct parasitological test as the gold standard method for CVL diagnosis. Accuracy was verified using the receiver operating characteristic (ROC) curve, which analyzes the performance of sensitivity and specificity data through the area under the curve (AUC) ranging from 0 to 1; values closer to 1 represent better ability of the test to discriminate between sick and healthy patients (Zweig and Campbell 1993).

ROC curve analysis was also used to establish the cutoff clinical score that could discriminate between CVL-negative and CVL-positive animals. The GraphPad Prism 6.0 software (GraphPad Software, Inc., USA) was used to analyze clinical signs, and the ratio test for two independent samples based on the Pearson chi-square distribution was used. The kappa index was used to compare serological tests with the gold standard diagnosis, the direct parasitological examination. The index interpretation was based on the study by Rosner (2006).

The distribution of clinical signs observed in CVL-positive dogs using the MH protocol (TR-DPP + EIE-LVC) was represented through descriptive statistics as a percentage. Biological variables such as age (up to 2 years and over 2 years); clinical classification (asymptomatic and symptomatic); gender (male and female); and hair characteristics (short and long) were analyzed by comparisons of proportions using the chi-square test (independence test) to assess their influence over the CVL diagnosis proposed by the MH (TR-DPP + EIE-LVC). Statistical analyses were performed using BioEstat 5.0 software (Ayres et al. 2007).

Results

Prevalence of CVL

During July 2015, a serological sample survey for CVL was conducted with 288 dogs from the municipality of Camapuã. Of these, 35.76% (103/288) samples yielded positive results through the screening test, TR-DPP, and 13.54% (39/288) were seropositive using the EIE-LVC test. Considering these results and based on the protocol of the MH, the prevalence of CVL was 12.5% (36/288). Of the 39 EIE-LVC-positive dogs, 3 had negative results using the immunochromatographic test, TR-DPP. However, direct parasitological examination provided a real CVL prevalence of 19.10% (55/288) in the municipality (Table 1).

Table 1.

Number and Percentage of Positive Animals Tested Through Direct Parasitological Diagnosis, Dual-Path Platform Immunochromatographic Test (TR-DPP), and Enzyme-Linked Immunosorbent Assay (EIE-LVC) and Respective Classification as Symptomatic or Asymptomatic Cases According to Silva Et Al. (2017) (n = 288)

Diagnostic test	Symptomatic animals	Asymptomatic animals	Total positive animals in the test
TR-DPP^®	30	73	103 (35.76%)
TR-DPP+EIE-LVC	13	23	36 (12.5%)
TR-DPP+direct parasitological examination	10	23	33 (11.46%)
EIE-LVC	13	26	39 (13.54%)
EIE-LVC+direct parasitological examination	6	19	25 (8.68%)
Direct parasitological examination	16	39	55 (19.10%)

Clinical assessment

According to the score system proposed by Silva et al. (2017), among the total of 288 dogs, 60 (20.83%) were classified as symptomatic (score >3) and 228 (79.17%) as asymptomatic (score: 0–3). Among dogs diagnosed with CVL, most animals were asymptomatic regardless of the diagnostic test applied: direct parasitological examination (70.9%), TR-DPP (70.87%), or EIE-LVC (66.66%) (Table 1). Therefore, it was not possible to establish an accurate threshold value of clinical scores, which could discriminate between CVL-positive and -negative dogs (Table 2). The direct parasitological examination is considered the gold standard methodology to diagnose leishmaniasis in animal or human patients. The diagnosis of VL is made by a combination of clinical signs, epidemiologic factors, and laboratory diagnosis. Laboratory diagnoses include a positive parasitology result with demonstration of the parasite or its DNA and detection of elevated titers of antibodies (Paltrinieri 2010, World Health Organization 2020). Since the serological tests performed in the study did not consider antibody titration, direct examination of lymph node aspirates was considered the gold standard test for serological comparison through the kappa index.

Table 2.

Sensitivity and Specificity of Cutoff Points to Distinguish Animals Positive for Canine Visceral Leishmaniasis Diagnosed Using Direct Parasitological Examination (n = 55 Animals) from Dogs Negative for Canine Visceral Leishmaniasis with Enzyme-Linked Immunosorbent Assay (EIE-LVC), Dual-Path Platform Immunochromatographic Test (TR-DPP), and Direct Parasitological Examination in Camapuã (n = 162 Animals)

Score	Sensitivity (%)	Specificity (%)
1	42.6	77.4
2	63.0	62.3
3	75.9	49.1
4	85.2	30.2
5	88.9	17.0
6	92.6	11.3
7	93.2	11.3
8	95.1	11.3
9	96.9	11.3
10	96.9	5.7

The most frequent clinical sign observed among EIE-LVC-positive dogs was enlarged lymph nodes (50%); followed by alopecia (30.56%); bad/opaque bristles (30.56%); ear lesions (25%); onychogryphosis (19.44%); muzzle ulcers/lesions (16.67%); pale mucous membranes (13.89%); weight loss (13.89%); muzzle depigmentation (13.89%); ocular discharge (8.33%); exfoliative dermatitis (8.33%); lip depigmentation (8.33%); lip lesions (5.56%); and blepharitis (2.78%). Apathy, bleeding, and congested mucous membranes were not observed in any evaluated dog.

Diagnostic test assessment

Table 3 demonstrates the performance of serological tests compared with direct parasitological examination. TR-DPP presented low sensitivity (0.62) as a screening test, while EIE-LVC showed high specificity (0.94) as a confirmatory test. Nonetheless, the combination of TR-DPP followed by EIE-LVC, as recommended by the MH (Brazilian Ministry of Health 2017), showed disappointing sensitivity (0.42) to diagnose CVL in an area with sporadic VL transmission.

Table 3.

Performance of Immunodiagnostic Methods Using Direct Parasitological Examination as the Gold Standard to Diagnose Visceral Leishmaniasis in Dogs From the Urban Area of Camapuã, Mato Grosso do Sul, Brazil, July 2015 (n = 288)

Test	Classification	S	Sp	PPV 95% CI	NPV 95% CI	AUC 95% CI
TR-DPP^®	General	0.62	0.70	0.33	0.89	0.66
	Asymptomatic	0.61	0.74	0.33	0.90	0.67
	Symptomatic	0.62	0.54	0.33	0.80	0.58
EIE-LVC	General	0.44	0.93	0.61	0.87	0.68
	Asymptomatic	0.46	0.96	0.69	0.90	0.71
	Symptomatic	0.37	0.84	0.46	0.79	0.60
TR-DPP+EIE-LVC	General	0.42	0.94	0.64	0.87	0.68
	Asymptomatic	0.43	0.97	0.74	0.89	0.70
	Symptomatic	0.37	0.84	0.46	0.79	0.60

AUC, area under the curve; CI, confidence interval; NPV, negative predictive value; PPV, positive predictive value; S, sensitivity; Sp, specificity.

For ROC curve analysis, the AUC demonstrated better performance for asymptomatic animals, using TR-DPP or EIE-LVC, solely or combined.

According to Rosner (2006), TR-DPP (kappa = 0.24) demonstrated reasonable agreement, while EIE-LVC (kappa = 0.42) and the combination of both serological tests, TR-DPP + EIE-LVC, presented moderate agreement (kappa = 0.42).

Biological variable analysis

Variables related to the biological characteristics of animals have been analyzed, but there was no significant influence on the results of TR-DPP + EIE-LVC, according to the protocol recommended by MH (Table 4).

Table 4.

Distribution of Biological Characteristics Observed in Canine Visceral Leishmaniasis-Positive Dogs with the Enzyme-Linked Immunosorbent Assay (EIE-LVC)

Biological characteristics	N	EIE-LVC+ (%)	χ² Value	p
Age
≤2 years	148	16 (10.8)	0.617	0.542
>2 years	140	20 (14.3)	0.617	0.542
Clinical classification^a
Asymptomatic	228	23 (10.1)	4.279	0.063
Symptomatic	60	13 (21.7)	4.279	0.063
Gender
Male	126	12 (9.5)	1.420	0.311
Female	162	24 (14.8)	1.420	0.311
Haircoat features
Short	218	32 (14.7)	3.161	0.117
Long	70	04 (5.7)	3.161	0.117

According to the score system proposed by Silva et al. (2017).

Discussion

A diagnostic screening tool for CVL should have high sensitivity to detect possibly infected animals that can act as parasite sources for the vector and increase the number of canine and human cases (Laurenti et al. 2013, 2014). Our results revealed a disappointing sensitivity value (0.62) for TR-DPP, therefore the prevalence of CVL was underestimated when the protocol of TR-DPP + EIE-LVC was applied in Camapuã. This result should be alarming for epidemiological control since many positive animals will not be detected and removed from the houses. Therefore, in regions with sporadic VL transmission, the serological protocol suggested by the MH seems to be insufficient to provide data for planning effective surveillance and control measures.

On the other hand, the immunochromatographic test did present a high negative predictive value (NPV) (0.89), as expected in low prevalence disease scenarios. Our data showed that the NPV was high even in the asymptomatic population (0.9). Asymptomatic dogs tend to be underdiagnosed through serological assays due to their predominant TH1 cellular immune response, with low antibody production, therefore resulting in false negative serological results (Solano-Gallego et al. 2011).

Hence, we reinforce the usefulness of TR-DPP as a confirmatory tool, despite its low sensitivity, due to the test's potential to segregate truly disease-free animals that can be kept in nonintense VL transmission areas, reducing the spread of the parasite. Laurenti et al. (2014) also proposed the use of TR-DPP as a confirmatory method because of its higher specificity when compared with EIE-LVC (Bio-Manguinhos) in their study. Furthermore, Coura-Vital et al. (2014) also recommended this rapid test as a confirmatory technique due to its ease of application and lower costs in comparison with EIE-LVC. It is also observed that the low sensitivity can be explained by the selection criterion since the direct parasitological diagnosis was the gold standard. This is used as a reference to identify truly positive animals using the epidemiological survey carried out by the municipal health surveillance service.

Usually, symptomatic dogs are easier to detect through serological evaluation since the expression of clinical signs correlates with the Th2 humoral immune response and positive results in diagnostic tests (Quinnell et al. 2001, 2013, Silva et al. 2017). Even in the present study, we notice a higher AUC to segregate asymptomatic dogs in both TR-DPP and EIE-LVC and their combination. This is an important characteristic for a diagnostic test in nonendemic areas because asymptomatic dogs usually represent the majority of infected canines in these regions (Riboldi et al. 2018), as demonstrated in Camapuã.

Silva et al. (2017) claim that rating a dog's clinical score using their methodology can provide veterinarians more confidence to diagnose CVL in areas with scarce diagnostic resources. These authors performed their study in an area of intense VL transmission in Brazil, at Teresina, Piauí. They suggested a cutoff value of ≥6 to distinguish CVL-positive animals from CVL-negative animals. However, this methodology might not be applied in sporadic transmission regions. Our results demonstrated that this approach would be unsuccessful in Camapuã since about 63% (23/36) of the dogs classified as CVL positive by the MH's protocol scored less than or equal to 3 points. Therefore, at Camapuã, it was not possible to determine an efficient cutoff value to identify CVL-positive animals using the Silva et al. (2017) scoring system.

Even though most of the positive animals (TR-DPP + EIE-LVC) were classified as asymptomatic, the clinical abnormality most seen in these dogs was lymph adenomegaly. Although it is a nonspecific clinical manifestation that does not raise high suspicion of leishmaniasis since it is present in many other canine pathologies, lymphadenopathy has been associated with higher parasitemia and infectivity in former studies (Verçosa et al. 2008, Laurenti et al. 2013).

Regarding serological diagnosis of CVL in asymptomatic dogs, Laurenti et al. (2014) have also observed higher sensitivity values of TR-DPP to diagnose the disease in these animals compared with symptomatic ones. The authors justified their results by suggesting that dogs involved in their study could be in an active state of infection, progressing to disease. This argument could also be applied in the studied population since animals were selected by stratified sampling using conglomerates and none of the biological features analyzed (age, clinical classification, gender, and haircoat) influenced the diagnostic tests.

Anyhow, it is important to emphasize that a different vector species of sand fly is involved in the transmission of VL in the studied municipality. Lu. cruzi is the probable vector of L. infantum in Camapuã (Oliveira et al. 2017a,b; Fernandes et al. 2017), while Lu. longipalpis is responsible for transmission of the parasite in the majority of the country (Aguiar and Medeiros 2003). Therefore, further investigations should be performed in this area because a different vector could result in L. infantum intraspecific strain variability and therefore raise distinct immune responses and clinical manifestations in canine hosts.

Vector and Leishmania species as well as geographical and epidemiological differences should also be considered when analyzing asymptomatic animals' importance as parasite reservoirs (Molina et al. 1994, Verçosa et al. 2008). Laurenti et al. (2013) demonstrated the high capacity of asymptomatic dogs from a Brazilian endemic area to transmit L. infantum to Lu. longipalpis through xenodiagnosis, while Travi et al. (2001) did not observe transmission involving the same vector and parasite species in asymptomatic dogs from an endemic area in Colombia.

On account of the exposed facts, further investigation of different clinical profiles of dog infectivity to the vector Lu. cruzi is necessary to better understand VL epidemiology in Camapuã and other similar epidemiological regions.

Conclusions

Canine leishmaniasis infection in nonendemic areas might have different characteristics from what is usually observed in endemic regions. The present study exemplifies this in a Brazilian area of sporadic transmission of VL. In Camapuã, the MH protocol is ineffective in estimating the prevalence rate and control measures. Besides, veterinarians must be alert even for asymptomatic dogs, especially those with lymph adenomegaly, since most animals will not present the classical expected signs of CVL, such as onychogryphosis and ear/muzzle lesions. Therefore, the combination of these two kits is not adequate for the diagnosis of CVL in an area of sporadic transmission as these two serological kits compared with microscopy have 40% sensitivity.

Footnotes

Acknowledgment

The authors are grateful for the support provided by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)—Finance Code 001.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This work was supported by Universidade Federal de Mato Grosso do Sul-UFMS-MEC, Brazil.

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