Study design
We conducted a cross-sectional, community-based study on volunteer participants by means of abdominal US. Participants were also interviewed using a structured and standardized questionnaire focused on potential risk factors and habits, including frequencies of their acting, which may favor infection (Supplementary material Annex I).
Work area
The study areas were the towns of Ñorquinco and Ramos Mexia as well as their surrounding rural areas in the Rio Negro Province (Figure 1), with a population estimated in nearly 3,200 people (1,800 in Ñorquinco and 1,400 in Ramos Mexia). Since the population census is more than 10 years old, the population was estimated by the primary health care service of the local hospitals, based on records arising from home visits by rural health workers. The majority of the population lives in urban areas. The rural population resides in small clusters (27 inhabitants in Treneta) or dispersed settlements around Ñorquinco (511 people).
Both towns have a small rural hospital staffed by two general practitioners, who coordinate the rural Primary Health Care Centres (PHCC) and the medical care posts (Figure 1). This health care network is the only health care provider in each area.
Ñorquinco area is located in the western mountain region of the province, extending over approximately 5,706 km2, with ideal conditions of humidity, temperature and vegetation for the survival of Eg eggs. Ramos Mexia area extends over approximately 9,680 km2, and is located in the east of the province, in the Patagonian steppe, where the very dry and hot summer could limits the survival of Eg eggs. The study areas have the conditions for the maintenance of the parasite life cycle: a high proportion of population with a low socioeconomic status, high number of families that own several dogs, a predominance of sheep farming for wool production, and the practice of home slaughtering sheep or goats and feeding dogs with raw viscera [8,11].
Both areas are targeted by the CE Control Program since 1980 in Ñorquinco and 1986 in Ramos Mexia; rural health workers are responsible for health education during house-to-house visits and for the distribution of praziquantel for dogs deworming four times a year. Deworming is usually demanded to the dog’s owner. Veterinary teams are responsible for the surveillance systems of the infection in dogs (originally with arecoline test and currently with coproELISA) and in sheep (by necropsy). Human surveillance includes doing regularly US screenings in schoolchildren performed by general practitioners and the systematic registration of cases identified [5,7,8]. As a result of program activities during the period 1980-1996, 1,720 new cases were identified in the province (an average of 101 cases per year), while in 2006-2016 period there were registered 478 cases (an average of 43 cases per year) [5].
Ñorquinco always presented higher prevalence rates than Ramos Mexia. For example, in 2003 the percentage of sheep farms with infected dogs (tested with coproELISA test / PCR) was 11.8% in Ñorquinco and 0% in Ramos Mexia, while the prevalence rate in schoolchildren (detected with US) was 1.0% in Ñorquinco and 0.3% in Ramos Mexia [5]. In both areas, prevalence shows a decreasing trend [8,12].
Population screening
Health workers from each rural hospital made house-to-house visits to explain the aim of the study and invite the people to participate as volunteer. US screening was conducted on a convenience sample of all volunteers including of all ages and both sexes , who agreed to participate. Each adult participant, or a parent or legal representative in case of minors, signed the informed consent form and filled the questionnaire (Supplementary material, Annex 1). Two US examination machines were installed in the hospitals (one or two US machines) and the others were rotated through the rural PHCC and schools. In total, 5 and 6 machines were available in Ramos Mexia and Ñorquinco, respectively.
The Focused Assessment with Sonography for Echinococcosis (FASE) protocol[13] was used for the abdominal US screenings. CE diagnosis and cyst staging was carried out according to the WHO Informal Working Group on Echinococcosis (WHO-IWGE) expert consensus [14]. CE case definition and clinical management were applied according to the Provincial Norms of Diagnosis and Treatment of Cystic Echinococcosis, approved by the Resolution 2624-2018 of the Ministry of Health, Rio Negro Province[5,13]. Briefly, in this document, a CE case is defined as the presence of pathognomonic features on imaging, or macro- or microscopic visualization or identification of any component of the CE cyst in a specimen, or morphologic changes of the cyst or seroconversion after medical treatment. A suspected CE case is defined by the presence of only one serological positive test (different than Western blot) or in the presence of a cyst without pathognomonic features on the image. In case of uncertain diagnosis, the participants were referred to the hospitals for further advanced imaging testing by US, CT, or MRI, as needed to finally classify them as confirmed or not, and then, the epidemiological information was processed according to the final diagnosis. All CE identified and confirmed cases were entered in the CE Control Program database and medical records were checked to verify whether the case was new or had a previous diagnosis of CE.
Data collection
The epidemiological information was collected using a standardized questionnaire (Supplementary material Annex 1), before the abdominal US scan was done.
The information collected demographic data (age, sex, place and time of residence, place of living during the first 5 years of life) health information (having a relative with CE in the house), behaviors associated with probable ingestion of Eg eggs (own and touch dogs, grow and eat raw and unwashed vegetables, source of drinking water, habits related to hand wash, smoke, use toothpick, and nail bite).
Analysis of data
The database was created in Microsoft Excel® 2.0 (Redmond, USA). A descriptive analysis of the variables was performed with EPIDAT 3.2TM (Xunta de Galicia, Spain) estimating the CE prevalence according to age, (sex, place of residence, and proportion of newly diagnosed and known and registered cases, with corresponding 95% confidence interval (CI). Place of residence during the first 5 years of life was classified for data processing in urban or rural areas and the years living in the area of the current address was categorized as less than 5 years, 5 to 10 years, 11 to 20 years and more the 20 years.
Bivariate analyses and multivariate analysis using STATATM 12.0 were used to estimate the Prevalence Rate Ratio (PR) with 95% CI of the association between CE and the variables studied.
The variable eat raw unwashed vegetables, was not included in the results because cases stated they cook the vegetables or always wash them.
A complete case approach for the multivariate analysis was used. A multivariate analysis binomial regression model was conducted starting with all factors that had a p-value =< 0.25 in the bivariate analysis and those risk factors that we decided a priori, based on existing published evidence model (dog ownership in the past 5 years and touching dogs). A manual stepwise backward selection was used to the final model. We used the lowest BIC (Bayesian information criterion) to identify the best-fitting models given the data collected. Only variables yielding a two-tailed p-value <0.05 was considered significant and presented in the final model