Introduction

CE or hydatid cyst is a zoonotic parasitic infection, caused by larval stage of cestode, member of the genus Echinococcus [1]. Currently, six species of Echinococcus have been identified, but the member of greatest clinical relevance and geographic distribution is Echinococcus granulosus (E. granulosus) [2]. The life cycle of this parasite includes carnivores (dogs and wolves) which represent definitive hosts and farm animals (sheep and cattle) which are intermediate hosts. The humans constitute the aberrant intermediate hosts [3]. Larval stage of parasite is located in the small intestine of mammals. Every day the adult cestode produces thousands of eggs, which are released through the faeces of carnivores into the surrounding environment. Eggs can be infectious for months or up to a year depending on the environment conditions. When the intermediate host ingests the eggs, they hatch, forming the oncosphere. It crosses the intestinal mucosa and reaches the internal organs such as the liver and lungs through the bloodstream. If the liver filter is passed, the parasite can reach the lungs, which represent the second site of definitive localization. If the lung barrier is also overcome, the parasite can reach and develop in any district of the body. The definitive host is infected through the ingestion of organs of the infected intermediate host. After ingestion, the parasite evaginate and attach the mucosa of its small intestine. Here the parasite develops until it reaches the adult stage after 30 - 60 days. The most common form of transmission to humans is the consumption of water or food contaminated with the faeces of infected carnivores [4-6]. The main risk factors are human contact with infected dogs and farm animals, rural lifestyle and lack of public health education [7]. The primary stage of infection in humans is manifested by an asymptomatic course, with well encapsulated cysts, which expand slowly and often go unnoticed for years [8]. The symptoms are related to several factors, such as: i) the number and size of the cyst; ii) location of the cyst within the affected organ; iii) the pressure exerted by the cysts on the adjacent tissues and structures [9]. In addition, the sudden onset of symptoms may be related to cyst rupture, leading to anaphylactic shock [10]. CE represents a socio-economic problem for humans and farm animals in different countries of the world [11]. Currently, CE have shown a prevalence of 2-3 million cases and an incidence of 200000 new infections per year worldwide [12]. The highest incidence of CE in humans and farm animals has been registered in Central Asia, northern and eastern Africa, Australia and Mediterranean regions [13]. This disease is endemic in Italy, especially in the southern regions. The reported prevalence of CE in southern Italy varies from 2.33% to 9.77% in the different regions [14]. Furthermore, it has been estimated that the impact of the disease, in terms of Disability Adjusted Life Years (DALYs), it is around 871000 with an annual cost of 3 billion US$ accounting for human treatment and livestock production losses [15]. Despite this strong socio-economic impact, CE is considered one of the 17 Neglected Tropical Diseases (NTD) by the World Health Organization (WHO) [16]. European Centre for Disease control (ECDC) has highlighted that Italy lacks a surveillance system for CE and, until few years ago, no official data are transmitted to European authorities (EFSA 2013) [17]. Since the beginning of the 2000s, the Campania Region has addressed the problem of parasites in organic and planned way, in particular with zoonotic aspect. With the aim of outlining the regional epidemiological framework and the role of some animal species in parasitosis chain, our region has been activated a series of regional projects. The land survey covered the Campania livestock population consisting of cattle, buffalo, sheep and goats. Surveys were carried out to assess the prevalence of Echinococcosis/Hydatidosis in sheep and goats’ farms. The actual spread of the CE in humans is not easy to define for the latency (often long) of the infection, before cyst detection, and for discontinuous information flows. The aim of the present study was to assess the current status of human CE epidemiology in Campania region, focused on the spatial distribution and epidemiological dynamics of this endemic zoonosis.

Materials and Methods

Study Design

Our work represents medical component results of regional project named: “Control and reduction of Echinococcosis/ Hydatidosis in animal populations and prevention of related human pathology - EchinoCamp1000”. The operating units of this project have been the Regional Center for the Monitoring of Parasitosis / Department of Veterinary Medicine and Animal Productions, University of Naples “Federico II “ (CREMOPAR), Zooprophylactic Institute of Mezzogiorno (IZSM) and the medical component made of Clinical Microbiology, University of Campania “Luigi Vanvitelli”. The current study was conducted from 2014 to 2018 in 650 farms, located in four provinces (Salerno, Avellino, Caserta and Benevento) of the Campania region in southern Italy. Geographic Information System (GSI) was used for analysis of study area. The aim was delaine the epidemiological aspect of Echinococcosis/Hydatidosis in Campania region to prevent the related human pathology. In view of the human severity disease, the early diagnosis of the cyst/s presence avoid surgery practice and allow the admission of drug therapy. In this regard the project has offered farmers, their families, and farm workers the possibility of CE serological and clinical diagnosis. Laboratory and clinical diagnosis were carried out. The laboratory diagnosis was performed through the use of two serological methods: Enzyme-Linked Immunosorbent Assay (ELISA) and Indirect Hemagglutination Test (IHA). These techniques have a good specificity and sensitivity; however, it is possible to find false positives in the case of subjects with neoplasms or other parasitosis [18]. As a result, they make it necessary to use a confirmation investigation, represented by the Western Blot (WB) [19]. While the clinical diagnosis provides ultrasound examinations for further confirmation to the patient’s clinical/surgical management. In the first project phase, the positive serological cases, in farms with suspected cases of zoonosis infection, were directed to instrumental diagnostic-clinical path at our University Hospital. In light of this finding and for the importance of related human diagnosis, the second-phase, in positive sheep farms, has included diagnostic research with ultrasound portable machine (MyLab™ Omega Esaote, MyLab). In this way was ensured complete human diagnosis avoiding follow-up losses in positive or potentially positive subjects.

Samples Collection and Analysis

In our study, we have collected 1497 peripheral blood samples for laboratory diagnosis. The samples were centrifuged, using a benchtop centrifuge (ThermoFisher, Massachusetts, USA), and the serum was separated and stored at -20 °C until analysis. All serum samples were screened by a commercial ELISA (Cypress Diagnostic, Langdorp, Belgium) and IHA (Dade Behring, Newark, NJ, USA), according to the manufacturer’s instructions. Samples that were positive or borderline to previous tests had been confirmed through WB (Euroimmun Labordiagnostika, Luebeck, Germany), according to the manufacturer’s recommendations. Seropositivity was determined when the WB result was positive.

Ultrasound

Positive participants were subjected to ultrasound examinations for hydatid cysts at University Hospital “Luigi Vanvitelli” of Naples. The ultrasounds were performed in the first phase at our University Hospital. In the second phase an internist doctor, with significant experience in the diagnosis of cystic echinococcosis, has been appointed with ultrasound portable machine. Ultrasound images were resolved according to WHO Informal Working Group On the Classification of Echinococcosis (WHO-IWGE). This classification ranks liver cysts into Cystic Lesions (CLs) and cystic echinococcosis in stages 1-5 (CE1-CE5). CE1 and CE2 represent active disease stages. CE3a is considered transitional disease stages. At the last CE4 and CE5 corresponds to inactive disease stages.

Data Collection and Analysis

The process of informed consent occurs with all the study participants that gave their authorization and agreement. Data and results were statistically analyzed by SPSS software (version 22.0, Chicago, IL, USA) [20].

Results

In the current study, 650 farms were visited, in particular 168 in Avellino, 123 in Benevento, 102 in Caserta and 257 in Salerno (Table 1). On the basis of the regional livestock population, cattle, sheep and buffalo of breeding farms were selected in several municipalities across the study area [21]. Analysis, performed by Geographic Information System (GSI), showed the distribution of farms in the region with small cluster in the south-eastern and north part of the study area (Figure 1) [22]. For each province, the distribution of the farms is shown in figure 2. A total of 1497 farmers were part of our study: 23,7% from Avellino, 18,0% from Benevento, 19,0% from Caserta and 39,3% from Salerno. This study population was composed most of male (61,5%) than female and regarding age distribution, the most representative was over 40 years (64,3 %) (Table 2). Peripheral blood sample were collected for each farmer. The serological ELISA and IHA screening detected 38 potential positive samples (2,5 %) out of 1497 blood specimens. Immunoblotting analysis confirmed 4 positive samples (0.4%) of which 3 in Salerno and 1 in Avellino (Table 3). All positives were found in over 40 years’ age group and 3 of them were male. CE was diagnosed in individuals, located in sheep farms (0,6%). The positivity to the echinococcosis through the serological screening has allowed to identified subjects without symptoms or confirmed the positivity of those already undergoing previous pharmacological or surgical treatment. So that, perform an analysis of the risk factors, like ultrasound examination, could be indicative of the human transmission of this parasitic infection. The serological positive participants were subject to ultrasound examinations for hydatid cysts. We reported a liver cyst detected by ultrasound portable machine diagnosis in one participant from Salerno province. The cyst was classified as CE3a stage (Figure 2).

Discussion

CE represents a neglected zoonotic parasitic disease that affects humans and livestock [23]. This disease is frequently caused by E. granulosus, species of greatest clinical relevance and geographic distribution [8]. CE determines serious socioeconomic and public health damage worldwide [24]. Over the past two decades, several studies focused on the global CE prevalence, in order to better define the endemic areas [13]. In contrast, few studies on CE epidemiology were conducted in Italy [14]. The purpose of this study was to evaluate the current state of human CE epidemiology in Campania region with the aim to build a surveillance network for this illness. In this regard has been implemented the disclosure activity to doctors and members of civil society with a focus on medical aspects. Surveillance of humans CE is difficult, mainly due to the asymptomatic infection in dogs and livestock and the long latency of parasite before the detected human cysts [25]. Study area involved 650 farms, spread over four provinces of Campania: 168 in Avellino, 123 in Benevento, 102 in Caserta and 257 in Salerno. We visited cattle, sheep and buffalo farms in the several municipalities of the study provinces. A total of 1497 peripheral blood samples were collected by each farmer and subjected to laboratory analysis. In this study, the diagnosis was performed through ELISA and IHA screening and WB confirmation. The serological screening found 2,5% of potential positive samples. WB analysis confirmed the 0.4% of positive cases of which 0,3 % in Salerno and 0,1 % in Avellino. Our results confirmed that the CE monitoring by ELISA and IHA overestimated the human exposure to E. granulosus in environments where contact with other helminths can occurs [26]. As proposed by WHO, the implementation of a confirmation technique had allowed to eliminate the false positives findings due to the cross reactivity with other parasites [27]. In our region, information and awareness campaigns to control echinococcosis through health education programs and information on disease severity and transmission modalities have contributed to the reduced prevalence of CE. Our findings showed a lower prevalence than observed in Iran (5%), Kenya (3.8%) and China (3.2%) [26- 28]. Differences in CE prevalence in several areas could be caused by: i) different climate that affects the viability of the parasite’s eggs; ii) the type of livestock farms in each country; iii) the role of the study population; iv) CE surveillance and monitoring systems [29]. Most of positive patients were males and belonged to over 40 years’ age group. These results were probably due to currently contact with farm animals, when cleaning or feeding them or during milking [30]. Positive cases possessed sheep farms (0,6%). Sheep are an intermediate host with high reception. In their livers and lungs, hydatid cysts mature easily and become potentially infectious to dogs and consequently to humans [31]. All serological positive participants were subject to ultrasound examinations for hydatid cysts. Ultrasound represent an effective, safe and inexpensive method that allows the recognition of this disease even in the asymptomatic phase [32]. According to WHO-IWGE, the ultrasound images of the patients were interpreted. This study, showed liver cyst in one participant (Salerno), classified as CE3a type. This cyst, detected by ultrasound portable machine, was in transitory stage, where the integrity of the cyst was compromised, producing the typical “water lily” sign [33]. Our study is the first that underline the epidemiological scenario in Campania region for the endemic human CE. Prevalence of human CE remains difficult, costly and time-consuming. For this reason, early diagnosis of cyst/s with ultrasound portable machine may prevent the routine surgery for CE and can offer to farmers and relatives a simple clinical-diagnostic path. It is now widely demonstrated that CE have a serious impact on welfare animal health, quality and quantity of production and also on professional’s health. In this scenario, the active collaboration between all the clinical figures involved, can help define a control common program for micro, small and medium-sized enterprises workers with the goal of testing, transferring and disseminating innovative techniques to detected this parasitosis.

Acknowledgement

We would like to show our gratitude to Campania Region that supported the project: “Control and reduction of Echinococcosis/ Hydatidosis in animal populations and prevention of related human pathology - EchinoCamp1000” and to all the partners of the project who provided insight and expertise that greatly assisted the research.

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