Current situation of the presence of the zoonotic nematode Dirofilaria immitis in dogs and humans in Bucaramanga, Colombia

Background: Cardiopulmonary dirofilariosis caused by Dirofilaria immitis is a zoonotic, vector-borne infection, whose main hosts are both domestic and wild canids and which can be transmitted to humans. It mainly occurs in tropical and subtropical climates, and temperature and humidity are the main factors that favor the presence and proliferation of Culicidae mosquitoes. There have been few reports of this disease in dogs and humans in Colombia, a region with favorable climatic conditions for the development of this disease. Therefore, this research aimed to study its current prevalence in dogs and the risk of human exposure to the disease in Bucaramanga, one of the most populated areas in Colombia located at the center of the country. Furthermore, its demographic and environmental characteristics could be useful as a study model for other similar locations and neighboring countries. Method: Serum samples from 351 dogs and 506 humans from the Bucaramanga metropolitan area were analyzed. All dog samples were tested with a commercial immunochromatographic test kit to detect the presence of circulating antigens of D. immitis. Human samples were analyzed using a non-commercial ELISA test kit to detect IgG against the somatic antigens of adult D. immitis and Wolbachia. Positive results were further confirmed using a Western blot analysis. Results: Dirofilaria immitis prevalence was 10.82% (95% CI) in dogs and 5.12% (95% CI) in microfilaremic dogs. Seroprevalence in humans was 6.71% (95% CI) and was significantly higher in individuals aged 16–34 years and in women than in men. Conclusions: This study describes seropositivity to D. immitis for the first time in a Colombian human population located in the same area as that of dogs infected with

3 D. immitis, which represents a potential threat to public health. In humans, age and sex can be considered risk factors for exposure to D. immitis. Background Cardiopulmonary dirofilariosis, caused by Dirofilaria immitis, is a vector-borne disease that occurs worldwide, and its main hosts are domestic and wild dogs and cats [1]. Different species of mosquitoes, such as the genera Culex spp., Aedes spp., and Anopheles spp., are involved in the transmission of this parasite. These species represent a constant risk of infection because they feed on both animal and human hosts [2]. Seroprevalence studies have been conducted in regions where infected dogs have been found, which indicated previous contact with the parasite and cases with pulmonary nodules. For this reason, dirofilariosis is considered an emerging public health problem because of its zoonotic potential [3,4].
Cardiopulmonary dirofilariosis is a chronic, progressive, and life-threatening disease. Adult worms are lodged in the pulmonary artery and the right ventricle of a dog's heart. Mosquitoes ingest the microfilariae after the females produce them, and after two successive molts, stage-3 larvae are inoculated into the definitive host during the next blood draw [2]. In humans, immature worms are embolized in the pulmonary microarteries, leading to the formation of benign lung nodules (pulmonary dirofilariosis), most cases of which are asymptomatic [1,2]. Moreover, D. immitis harbors endosymbiotic bacteria of the genus Wolbachia, which participate in the parasite's molting and embryogenesis and play a key role in the immune and inflammatory response to the disease [5,6].
Dirofilaria immitis is primarily located in tropical and subtropical climates and depends mainly on environmental factors, including temperature and humidity, in 4 addition to human behavior such as implantation of irrigation systems, taking pets on trips, and new urban developments, that favor the presence and proliferation of its transmission vectors. However, the number of reports in areas with less warm climates has increased, which indicates that the disease is expanding [1,2,7,8].
The South American continent is one of the most biodiverse areas on the planet, with a combination of factors such as intensification of agricultural practices, landscape modification, poor ecosystem protection, and potentially unstable economies, which potentially lead to the spreading of the disease and its vectors [9]. On this continent, the disease has only been reported in Argentina, Colombia, Peru, and Brazil, with relatively few studies in domestic dogs; very few sporadic cases of pulmonary dirofilariosis have been described [1,10,11,12]. The disease has been reported in dogs from different areas of Colombia, even in high-altitude areas with cold weather, with mean prevalence values of 0.91-16.12% according to different methodologies [10,13]. Only one case of human pulmonary dirofilariosis has been described [14], and two seroepidemiological studies were conducted in an area within the Colombian Amazon where infected dogs were also found [15,16].
The purpose of this investigation was to determine the presence of D. immitis in dogs and their possible contact with the human population in the metropolitan area of Bucaramanga, Colombia.

Sampling area
The metropolitan area of Bucaramanga, which belongs to the capital city of the Department of Santander in Colombia, includes the municipalities of Bucaramanga, Floridablanca, Piedecuesta, and Girón ( Fig. 1), located near the capital of Colombia, Bogotá. It extends to an area of 1,479 km 2 , and the municipal area occupies 165 km 2 ; it is located at 959 m above sea level. It includes two sectors of different geographical conformations: one formed by a plateau and the other by a valley. Its climate is tropical, with a mean annual temperature of 23.4 °C and significant precipitation levels with an approximate mean of 1,159 mm [17]. It has an estimated population of 1.2 million people, and there are 32,000 censored dogs in the city of Bucaramanga alone. In addition, there are numerous uncensored vagrant dogs throughout this area [18].

Samples used
This study included samples from 351 dogs and 506 humans collected during the months of February-June 2018. The dog and human samples were collected by members of the veterinary staff of different clinics and associations and the Higuera Escalante Laboratory's health care staff, respectively. For the canine population, signed informed consent from the owners was considered the inclusion criterion.
Variables considered for the analysis were age, sex, municipality of residence, socioeconomic status, and whether they lived inside or outside a house. For the human population, being of legal age and signing the informed consent were the inclusion criteria. Variables for the analysis were age, sex, municipality of residence, socioeconomic status, living with pets, type of pet (dog or other), and presence of water sources at < 200 meters. Confidentiality of patient information was always maintained, and all study participants gave their written consent.
The number of samples collected by age groups of < 1, 1-3, 4-6, 7-10, and 11- were considered positive when both non-commercial ELISAs were positive for the same serum sample. These results were additionally confirmed using Western blot analysis performed according to a previously described methodology [23,24]. Both antigenic extracts were subjected to SDS-PAGE in 12% gels under reduced 8 conditions, and proteins were transferred onto nitrocellulose. Human sera were analyzed at a 1:40 dilution and anti-conjugates at a 1:500 dilution.

Geospatial analysis
A geospatial analysis was performed on the population of dogs and humans from the metropolitan area of Bucaramanga through a spatial overlay of positive cases using the SatScan software v.9.6. and the Bernoulli's model with a 95% significance level (999 replications with p < 0.05) based on the Monte Carlo statistical significance test. Further, we established clusters, which are areas with a relative risk of infection in dogs and humans, with a maximum size of 50% of the exposed population, based on population census and positive cases. Clusters were imported into the QGIS software (3.8.0 version) to be visualized on the study area map.

Statistical analysis
Data was analyzed using SPSS 20.0 software for Windows (SPSS Inc./IBM, Chicago, IL, USA). This is a descriptive study with a univariate analysis for the determination of frequencies and a bivariate analysis through chi-square and OR estimation, based on which a statistical analysis was performed for the determination of the association between variables. In all cases, the level of significance was established with p < 0.05.
No statistically significant differences were observed between the variables sex, age groups under 7 and over 7 years, socioeconomic status, municipality, and sleeping inside or outside a house. However, a statistically significant relationship was observed between municipalities and presence of microfilaremia (p < 0.01). No association was observed between the variables sex, socioeconomic level, and municipality, but age of dogs and whether they lived inside or outside a house can be considered risk factors for exposure to D. immitis (OR = 1.75 and 1.48, respectively). No statistically significant differences were found between exposure to D. immitis and the other variables, and no association was observed between them (p < 0.05).
There were no statistically significant differences between the variables and exposure to the parasite. There was no association between the variables of living with pets or not, especially dogs, but age and sex can be considered risk factors for exposure to D. immitis in humans (OR = 1.29 and 1, 75, respectively).
In the spatial exploration of dogs positive for D. immitis within the study area ( Fig. 3), no significant clusters were found with p < 0.05. However, cluster 1 showed a relative risk of 7.90 in dogs. However, three significant clusters were detected taking into consideration the magnitude and distribution of seropositive humans:  (1,4,7,22,26,27,28]. In Colombia, dirofilariosis is a very poorly studied disease. Few studies have addressed this problem during the last 20 years [10,13,15]. Furthermore, prevalence values in dogs vary (0.91-16.12%) and have been obtained using different methodologies. In humans, there is only one clinical case of a patient from whom an adult worm identified as Dirofilaria sp. was extracted from the lung [14], and there are two studies that warn of the existence of human infections caused by D. immitis in communities from the Colombian Amazon where infected dogs have been found [15,16].
In this study, we analyzed the presence of D. immitis in dogs and humans in the 13 Regarding human infections, mean seroprevalence was 6.71% and the highest prevalence was 11.7% in Piedecuesta, followed by 6.9% in Bucaramaga, where prevalence in dogs reached similar values (12.5% and 7.6%, respectively); moreover, we observed slightly lower seroprevalence values of 5.7% in Floridablanca and 3.1% in Girón. where the prevalence observed in dogs was 13.7% and 12.9%, respectively. These data are related to the geographical location of the samples, and humans with positive serology have been reported in the same location as infected dogs. In addition, there are spatial clusters in these areas with a relative risk of < 1 for humans, suggesting a positive association between the variables studied and a higher frequency of contact with the parasite. Thus, these data suggest a direct relationship between the presence of dogs and humans infected by D. immitis. This is similar to what occurs in other European areas such as Spain, Portugal, Romania, and Russia, where the risk of infection among humans has been studied [4,7,8,27,28,29].
Regarding the variables evaluated, sex may be a risk factor for human exposure to D. immitis. In our study, we found that seropositive individuals were mostly women.
There are several studies that suggest that serological screening in humans should be carefully interpreted [7,8,28,29]. Further, we observed that age can be a risk factor. In our study, the population with the highest seropositivity was that from the age group of 16-34 years. However, other studies report that the risk of infection increases with age [4,7,8,27,28,29]. Humans within this age group perform more activities outside their homes and in areas with vegetation. However, it is necessary to analyze this fact more closely, and more data and optimization of serological screening are required in future studies.
Furthermore, not only did this study allow us to address the problem from a biological point of view but also from a socioeconomic point of view in case of humans. The highest seroprevalence was observed in stratum 1, where sanitary hygiene conditions are not adequate (20%), followed by stratum 2 (8.3%), stratum 3 (5.7%), and stratum 4 (4.1%). Seropositive individuals were not detected in the last two strata where the sanitary hygiene level is optimal. Socioeconomic status has been associated with mortality and the use of health services, which indicates that a lower income reduces the application of prophylactic and preventive measures to vectors and canines that live with humans (González de Haro, 2006). Environmental sanitation elements, such as water; sewage, garbage, and waste disposal; sanitary landfills; and garbage treatment, influence the prevalence of parasitosis. These data allow us to associate the lack of sanitary hygiene with the development of dirofilariosis, which may become a socially determinant public health factor, as in the case of other vector-borne diseases in Colombia such as malaria, leishmaniosis, Chagas disease, and human ascariosis [9,30,31].

Conclusions
In conclusion, this study describes, for the first time, seropositivity to D. immitis and WSP in a human population in Colombia, specifically in one of the most populated areas of the country, the metropolitan area of Bucaramanga, with a high presence of dogs infected by D. immitis, which are a potential threat to public health. The corresponding authorities should take measures to monitor and control this emerging zoonotic disease to reduce prevalence in canines, while including human pulmonary dirofilariosis in the differential diagnosis of pulmonary nodules.

Ethics approval and consent to participate
The sampling process complied with the Helsinki code of ethics and animal welfare and was approved under resolution by the Ethics Committee of Universidad Cooperativa de Colombia.

Consent for publication
Not applicable.

Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.

Competing interests
The authors declare that they have no competing interests.

Funding
This study has been supported by Universidad Cooperativa de Colombia (INV2213) and Agencia de Desarrollo Económico de Castilla y León, Spain (cofinanced with FEDER funds).

Authors' contributions
MVEM, RM and VAQ designed the study and wrote the manuscript. LAN, IH and CFA performed the fieldwork and collected the data. MVEM, RM and VAQ participated in the discussion of the results and corrected the manuscript All authors read and approved the final manuscript.

Acknowledgments
We would like to thank the staff of the Higuera Escalante Blood Bank and veterinarians from the companion animals' clinic of Universidad   Graphical Abstract.tif