Seroprevalence of Human Brucellosis among Syrian Refugees in Jordan, 2022

Introduction Brucellosis is prevalent in Mediterranean countries. The aim of this study was to determine the seroprevalence of brucellosis and associated factors among Syrian refugees in Jordan. Methods A cross-sectional study was conducted among adult Syrian refugees who attended the Public Health Lab (PHL) in Al Mafraq governorate, during the period of May-June 2022 to obtain a health certificate, which is legally required to receive governmental authorization for employment in Jordan. Blood samples were obtained from participants and a serum specimen was tested for the presence of IgG antibodies against Brucella using enzyme-linked immunosorbent assay (ELISA) IgG kits (Vircell Microbiologists, Granada, Spain). Results A total of 1562 Syrian refugees were enrolled in the study. Their ages ranged between 18 and 74 years, with a median age of 30 years at presentation. The majority were males (75.9%, n = 1186) and 24.1% (n = 376) were females. The Brucella ELISA IgG results were positive for 149 persons, with an overall seroprevalence rate of 9.5% (95% confidence interval: 8.0%–11.0%). Having animal-related occupations, residing outside refugee camps, consuming unpasteurized milk, handling animals or their tissues, and slaughtering animals within 6 months of study inclusion were significantly higher among the seropositive group. In the multivariate analysis, IgG-positive persons were 13 times more likely to report being diagnosed with brucellosis (OR = 13.1, 95% CI: 6.1–28.3; p ≤ 0.001). In addition, they were more likely to reside in the city of Al Mafraq, as opposed to a refugee camp (OR = 1.9, 95% CI: 1.1–3.2; p = 0.025) and to have handled animals within 6 months of study inclusion (OR = 3.1, 95% CI: 1.1–8.9; p = 0.035). Conclusions In conclusion, one-tenth of adult Syrian refugees were tested positive for Brucella ELISA IgG. Being diagnosed with brucellosis, residing in the city of Al Mafraq, as opposed to a refugee camp, and handling animals within 6 months of study inclusion were significantly associated with being positive for Brucella ELISA IgG. This study illustrates the need for improved brucellosis control measures via comprehensive vaccination of animals and enhanced laboratory detection and surveillance capacities, in addition to emphasizing the need for increased awareness sessions among Syrian refugees on the safe use and preparation of dairy products and safety practices of handling animals and their tissues.


Introduction
Brucellosis is a bacterial endemic zoonotic disease of global signifcance with detrimental impacts on public health and food animal production.It is mainly transmitted to humans either through contact with infected animals (typically small ruminants and bovines) or due to consumption of unpasteurized dairy products as bacteria sheds in milk and other discharges [1].In humans, brucellosis as an illness is debilitating, and its symptoms, including fever, sweating, fatigue, weight loss, headache, and joint pain may persist for weeks to months [2,3].In domestic animals, the disease causes signifcant livestock production losses resulting in major consequences on the economies of afected countries [4].Furthermore, Brucella is being increasingly recognized for its potential use as a biological weapon [5].
Te burden of the disease is more pronounced in developing countries due to inadequate surveillance and control measures, reduced domestic and imported animalbased control programs, and a lack of suitable diagnostic services.Te highest incidence rates of brucellosis have been reported in Mediterranean countries [6], including Jordan.However, there is little information on the risk factors and geographical patterns of human brucellosis in Jordan and its characterization among refugees.
In spite of the considerable progress of knowledge gained and success achieved in brucellosis control in the developed world, this disease continues to be an important burden in the Middle East [1].Over the last decade, an average of about 145 brucellosis cases was reported by the Ministry of Health (MOH) annually in Jordan, with the incidence rate of brucellosis almost doubling from the ten-year period of 2010 to 2020 (2.1 to 4.5 per 100,000 population) [1,7].However, the disease tends to be universally underreported [1].Interestingly, in all published reports, Karak and Al Mafraq governorates accounted for over 50% of reported cases, despite representing less than 9% of the total population of Jordan [1,7].
Data on seroprevalence in Jordan and Syria are limited and old.Te only study in Jordan was conducted in 1992 among high-risk individuals in the north of Jordan.Te study revealed a signifcantly higher seroprevalence of brucellosis among high-risk people (8.2%) compared to the control sample (0.5%).Seroprevalence among sheep farmers (12.5%) and meat handlers (4.9%) was signifcantly higher than that with other occupations [8].On the other hand, studies conducted in Syria revealed that the seroprevalence of brucellosis in cattle ranged from 17.48% in 1992 to 2.59% in 1996, due to vaccination campaigns [9].A nationwide cross-sectional study in Jordan showed that the estimated seroprevalence values were 18.1% (95% CI: 11, 25.3) in cattle herds, 22.2% (95% CI: 16.5, 28.8) in sheep focks, 38.5% (95% CI: 24.3-51.8) in mixed herds of cattle and small ruminants, 45.4% (95% CI: 30.3, 61.6) in goat herds, and 70.4% (95% CI: 55.5, 84.9) in mixed sheep-goat focks.Te true prevalence across all small ruminant focks was estimated as 34.3% (95% CI: 28.4,40.4) [10].Alarmingly, the World Organization for Animal Health's report revealed Syria to have the highest annual incidence rate of human brucellosis worldwide, reaching an annual rate of 1603 cases per million [11].
Te high incidence of brucellosis in Jordan is noteworthy because Jordan has undergone signifcant socioeconomic shifts due to the recent confict in the neighboring country and the consequent infux of around 1.3 million Syrian refugees since 2013 [12].Te confict has been associated with an increased incidence of infectious diseases [13], including brucellosis in neighboring or host countries.In fact, cases of brucellosis among Syrian refugees are being increasingly detected in European countries [14,15].No studies have yet been conducted to estimate brucellosis incidence among Syrian refugees of Jordan, but previous reports have highlighted the increased smuggling of unvaccinated livestock along the Jordan-Syrian border in recent years [16].In addition, the Al Mafraq Health Directorate's annual reports of human brucellosis cases within its governorate revealed that between the years of 2019 and 2021, an average of 132 cases were detected, of whom 38 patients were Syrian.
A cross-sectional study was conducted to estimate the seroprevalence of brucellosis and determine its associated factors among Syrian refugees in the Al Mafraq governorate in the north of Jordan, including residents of the governorate's refugee camp.Only those who had a verifable identity card proving their Syrian nationality and refugee status were eligible for inclusion.Te study team explained the purpose of the study to eligible subjects and a blood sample was withdrawn after they provided their written informed consent.

Methods
Assuming that the seroprevalence is 10%, the sample size needed to estimate the seroprevalence within a margin of error of 2% was calculated at 865 at a power of 80% and level of signifcance of 0.05.Te sample size was calculated using Statulator [18].

Investigation Form.
Te study participants were interviewed by using a pretested structured questionnaire that was adapted from the previously validated investigative forms published by the United States Centers for Disease Control and Prevention (CDC) [19] and the ofcial case report form of the Ministry of Health of Jordan.Te questionnaire included questions regarding the age, sex, education, employment, exposure to animals, history of symptoms suggestive of brucellosis, history of established brucellosis diagnosis, and description of treatment received, if any.Te investigation form included questions regarding certain characteristics or activities that have been reported as risk factors for brucellosis.Tese known risk factors included eating raw meat, consuming unpasteurized milk (or fresh nonboiled milk), consuming dairy products prepared from nonboiled milk, preparing dairy products at home, handling animals, animals' vaccination, handling animal tissues (placenta, aborted fetus, and birth fuids), and slaughtering/skinning animals within 6 months from the date of flling the investigation form.

2
Journal of Pathogens Te participants were asked if they experienced any of the following symptoms within 6 months of the date of flling the investigation form: undulant fever, night sweats, headache, joint pain, loss of appetite, malaise, myalgia, back pain, and depression.
Te KoboToolbox application (Kobo Inc., Ontario, Canada) was used to create a data entry screen with necessary validation rules and skips.Android-operated tablets loaded with Kobo software were used for data collection.Data were synchronized daily with a secure FTP server at the Eastern Mediterranean Public Health Network (EMPH-NET) ofces to allow for repairing any data entry errors while data collectors were still in the feld.

Lab Testing.
At the PHL, sera samples were separated daily upon arrival and kept at 2-8 °C until processed (within a maximum of three days of specimen collection), and each serum specimen was tested for the presence of IgG antibodies against Brucella using enzyme-linked immunosorbent assay (ELISA) IgG kits (Vircell Microbiologists, Granada, Spain).Universal biosafety and preventive precautions were considered during the withdrawal, transportation, and testing of blood specimens.
2.5.Data Analysis.Data analysis was conducted using SPSS version 24 (IBM, Armonk, New York, United States).Data were described using percentages.Te chi-square test was used to compare background and Brucella-related variables between the ELISA IgG-positive and the ELISA IgG-negative subjects.Binary logistic regression analysis was conducted to assess the associations between studied variables and the ELISA IgG results.A p value of less than 0.05 was considered statistically signifcant.

Participants' Characteristics.
A total of 1562 Syrian refugees were enrolled in the study.Teir ages ranged between 18 and 74 years, with a median age of 30 years at presentation.Te majority were males (75.9%, n � 1186) and 24.1% (n � 376) were females.Of those, 20 (5.3%) were pregnant at the time of the study.Te median family size was 5 members.Of all participants, 410 (26.3%) reported that they are currently employed.Of the employed participants, 78 (19.0%) reported having animal-or animal-tissue-related occupations such as shepherd, butcher, animal farm guardian, producing dairy products, working on poultry farms, and handling or packing meat products.Only two of the participants in this category were females (both working as farmhands on farms containing animals).When asked about their address, 303 (19.4%) of the participants revealed that they are residents of the Zaatari camp (Table 1).

Brucella-IgG Prevalence and Associated Characteristics.
Te Brucella ELISA IgG results were positive for 149 persons, resulting in an overall seroprevalence rate of 9.5% (95% confdence interval: 8.0%-11.0%).Of those, 21 (14.1%) reported being previously diagnosed with brucellosis, and 20 received treatment (rifampicin and doxycycline) after their diagnosis.Te reported length of treatment ranged between 20 days and 2 years (42 days was the most frequent response (n � 7)).Te ages of the seropositive participants also ranged from 18 to 74, with a median age of 33 years.
Overall, 38 (24.5%) of the seropositive participants were employed; 15 persons had animal-related occupations.Te proportion of persons who reported having animal-related occupations was signifcantly higher among seropositive subjects than in seronegative subjects (p � 0.003).Only 13.4% of the IgG-positive cohort resided in a refugee camp compared to 21.7% of the IgG-negative participants (p � 0.009).
When asked if they recalled being previously diagnosed with the disease, 14.1% of the IgG-positive cohort reported a history of brucellosis diagnosis compared to only 1.0% of the IgG-negative participants (p ≤ 0.001).In addition, 5.8% of the IgG-positive and 0.7% of the IgG-negative participants had family members who were diagnosed with the disease (p ≤ 0.001) (Table 1).
Overall, 26.8% (n � 40) of the Brucella IgG-positive participants reported at least one of these characteristics: eating raw meat, consuming unpasteurized milk or fresh nonboiled milk, consuming dairy products prepared from nonboiled milk, preparing dairy products at home, handling animals, handling animal tissues (placenta, aborted fetus, and birth fuids), and slaughtering/skinning animals within 6 months from the date of flling the investigation form, compared to 14.4% (n � 203) of the IgG-negative participants (p ≤ 0.001).Consumption of unpasteurized milk, slaughtering animals, and handling their tissues were signifcantly more prevalent among the IgG-positive participants (Table 1).

Multivariate Analysis.
In the multivariate analysis, IgGpositive persons were 13 times more likely to report being diagnosed with brucellosis (OR � 13.1, 95% CI: 6.1-28.3;p ≤ 0.001).In addition, they were more likely to reside in the city of Al Mafraq, as opposed to a refugee camp (OR � 1.9, 95% CI: 1.1-3.2;p � 0.025), and to have handled animals within 6 months of study inclusion (OR � 3.1, 95% CI: 1.1-8.9;p � 0.035) (Table 2). 3 shows the diferences in participants' responses to symptoms questions according to the IgG-status.Among the symptomatic participants, the average number of symptoms displayed within 6 months of data collection was 6 symptoms for the IgG-positive cohort and 4.4 for the IgG-negative cohort.Undulant fever, headache, joint pain, and loss of appetite were signifcantly more prevalent among the seropositive group.Overall, the portion of those who experienced any of the abovementioned symptoms was signifcantly larger among the seropositive participants (6.0% vs 2.8%, p � 0.045) (Table 3).However, multivariate logistic regression analyses of the reported symptoms revealed that none of the symptoms were signifcantly associated with a positive Brucella IgG result (Table 4).

Discussion
Tis is the frst study of brucellosis seroprevalence among a relatively large number of refugees in Jordan.Te fndings of this study portray the overall status of the seroprevalence of Brucella IgG among Syrian refugees in Al Mafraq and the governorate that hosts the largest population of Syrian refugees in Jordan, as well as containing the Zaatari camp.
Te results revealed that close to 10% of the population might have a past brucellosis infection as evidenced by the detection of Brucella antibodies in their sera.Tis rate is higher than the rates reported in other areas such as Jammu in India (4.96%) [20], Laconia in Greece (8.0%) [21], and the Wadi Al Dawasir region of Saudi Arabia (8.6%) [22].Te fndings of this study have signifcant implications for shaping government health policies on brucellosis.Te fndings highlight the need for enhanced surveillance and screening programs, not only for the refugee population but also for the host population.Te government may consider expanding its testing eforts to identify and treat individuals with brucellosis more efectively.Te government should evaluate and possibly expand its healthcare infrastructure to accommodate the increased demand for healthcare services resulting from a higher prevalence of brucellosis among the refugee population.
Te analysis revealed that some variables were signifcantly associated with ELISA IgG results, particularly concerning the nature of their occupations, their locations, habitual exposure to, and slaughtering of livestock, as well as drinking unpasteurized milk.
Unpasteurized milk has long been considered the primary factor for brucellosis in humans [1,2].While only 18 participants in our cohort reported recent consumption of unpasteurized milk, six participants (33.3%) had positive Brucella IgG results (p � 0.005).Consumption of raw or unpasteurized dairy products and consumption of raw or undercooked meat was not signifcantly associated with seropositivity in this study.On the other hand, the seropositive cohort of the study population was signifcantly likely to have handled animals, primarily small ruminants (sheep in 82% of the cases) (OR � 3.1, 95% CI: 1.1-8.9;p � 0.035).Tese observations corroborate the trend observed in previous reports; whereby for lower to mid-income countries, brucellosis is more of a public health concern in terms of direct contact with livestock, rather than being a food-borne concern, as is the case of industrialized nations [23,24].Tis implies that any preventative actions or awareness activities to be conducted in Jordan may beneft from targeting animal-handling and occupational safety protocols and not solely focusing on food safety recommendations.
In this regard, a review of Brucella seroprevalence data in Bangladesh demonstrated that it was highest among those who had direct contact with animals, its products, and those who consumed raw milk.Te seroprevalence was highest among livestock farmers (2.6%-21.6%),followed by milkers (18.6%), veterinarians (5.3%-11.1%),and butchers (2.5%) [25].Similarly, many of the participants in our study revealed that they had occupations that involved direct contact with animals and their fuids/tissues (n = 78, 19.0%), with the higher portion observed among the seropositive group (10.5% vs. 4.5%, p = 0.003).Most frequently, those with animal-related occupations had agricultural professions, such as being a farmhand, or shepherd/herder (23 and 9 participants overall, respectively).Tis is in agreement with previous reports on agricultural occupations being the major sector of employment among Syrian refugees in Jordan [26].Te fact that 19.2% of those with animal-related occupations were IgG-positive, in addition to the seropositivity of 41.1% of the participants who handled animals highlights a need for increased awareness of the population working in the agricultural sector in the country, as it is possible that they are exposed to the disease in their jobs, which was observed in previous studies [27].Interestingly, refugees living in refugee camps were less likely to be IgG-positive than the refugees living in the city of Al Mafraq and the governorate's neighboring towns, raising attention to the need for ensuring their inclusion in any brucellosis prevention, control, or awareness activities that may be conducted for refugees in the governorate.
Regarding the clinical presentation of the study participants, only 6% reported having any brucellosis symptoms within the last six months, with the rate being signifcantly higher among the seropositive group than that observed among the seronegative group.As Brucella IgG positivity is an indicator of a possible past infection or chronic case of brucellosis [28], seropositive participants might have exhibited the symptoms at an even earlier stage.Moreover, only 14.1% of the seropositive group recalled ever being diagnosed with the disease.Tis is a signifcant observation as it can be an indication that many of the brucellosis cases might have been misdiagnosed or undiagnosed.An investigation of 141, 604 lab-confrmed brucellosis cases in China by Wang et al. revealed that 57.6% of the patients had been misdiagnosed or suspected of having another disease, which was attributed to the absence of characteristic brucellosis symptoms and reduced awareness among physicians [29].
It is worth noting that the study has several limitations.For instance, the participants consisted of those arriving at the PHL of Al Mafraq on their own accord, which may have contributed to the relatively high portion of male participants (75.9%), in comparison to actual estimates that 51.6% of the overall population of Al Mafraq and 51% of the refugee population of Zaatari are males [30].Te study's focal points at the PHL reported that many of the participants came to the PHL to obtain a health certifcate, which is legally required to receive governmental authorization for employment in Jordan.Tis also applies to the 303 refugees arriving from the Zaatari camp, as the local regulations in Jordan require that all residents and refugees in Al Mafraq can only obtain their ofcial health certifcates (whether employment or travel) from the PHL, despite them having access to public health centers and lab facilities at their camp.Reports have revealed that a minority of female Syrian refugees in Jordan join the labor force [31].In our study, of the 332 female participants in this study, only 26 (7.8%) were employed.Future studies may thus beneft from utilizing a longer study duration, including a wider age group, and using an active sampling strategy to gain more comprehensive cohorts.
Nonetheless, as the frst report of brucellosis seroprevalence among a large group of Syrian refugees, this study may be considered a baseline for future investigation of human brucellosis regarding refugees in Jordan and other at-risk populations in the country.
In conclusion, one-tenth of adult Syrian refugees tested positive for Brucella ELISA IgG.Being diagnosed with brucellosis, residing in the city of Al Mafraq, as opposed to a refugee camp, and handling animals within 6 months of study inclusion were signifcantly associated with being positive for Brucella ELISA IgG.Our study implies a need for increasing awareness among both the general population and the healthcare providers of Al Mafraq to address the apparent inefcient diagnosis of brucellosis, especially considering the importance of timely diagnosis for efective treatment of the illness and prevention of chronic disease.Ultimately, our results illustrate the need for improved brucellosis control measures via comprehensive vaccination of animals and enhanced laboratory detection and surveillance capacities, in addition to emphasizing the need for increased awareness sessions among Syrian refugees on the safe use and preparation of dairy products and safety practices of handling animals and their tissues.

Table 1 :
Te demographic and relevant characteristics of participants according to Brucella IgG test result.

Table 2 :
Univariate and multivariate analysis of factors associated with Brucella seropositivity.

Table 3 :
Te diference in reported symptoms (within 6 months of the date of data collection) between the Brucella IgG-positive and IgG-negative cohorts.

Table 4 :
Univariate and multivariate analysis of signs and symptoms associated with Brucella seropositivity.