Summary
Introduction: Brucellosis causes a necrotic and inflammatory infection in humans and animals, and is among the world's most common zoonotic diseases. The aim of this study was to determine the seroprevalence of Brucella antibodies and compare serological methods in Turkish Republic of North Cyprus, where animal husbandry is common.
Materials and Methods: The study was conducted between December 2017 and February 2018 and included veterinarians (n=50), animal caregivers (n=109), butchers (n=65), and a control group of individuals who had no connection with animals (n=100). Serum samples from the participants were analyzed with serological techniques including Rose Bengal test (RBT), standard tube agglutination test (STA) and ELISA; IgG and IgM methods. The sensitivity and specificity of the methods used were estimated by considering ELISA (IgG and/or IgM) results as reference/golden standard.
Results: Of 27 patients (8.3%) patients who had positive results from at least one of the serological tests, 21 (6.5%) had positive RBT, 15 (4.6%) had positive STA and 10 (3.1%) had positive ELISA (IgG and/or IgM). Six (28%) patients with negative RBT results were found to be positive in ELISA. Seventeen samples (80.9%) were RBT-positive but ELISA- negative.
Conclusion: These data suggest that Brucella infection is at low rate in the Turkish Republic of North Cyprus. Laboratory diagnosis should be supported by tests such as seroconversion for low titers on STA, or tests such as ELISA. Only two cases were reported to the Ministry of Health Statistics Unit in 2013.
Introduction
In Cyprus, the prevalence of brucellosis in animals was claimed to be 0.1% since 2007 as a result of the eradication programs conducted at various time periods[1]. Brucellosis (in both animals and humans) is a notifiable disease in the Turkish Republic of Northern Cyprus (TRNC). According to TRNC Ministry of Health records, its prevalence in humans is on the rise. This increase has been attributed to improved diagnosis, a higher reporting rate, and inadequate disease control[5]. The aim of this study was to determine the seroprevalence of Brucella melitensis and Brucella abortus antibodies in TRNC, where livestock farming is widespread. The study included high-risk groups such as veterinarians, animal care workers, and butchers. Due to the lack of previous research on Brucella spp. in TRNC, we believe that this study will contribute to our national data.
Methods
The serum samples were analyzed using Rose Bengal test (RBT) (Pendik Veterinary Control and Research Institute, Turkey), standard tube agglutination test (STA) (Pendik Veterinary Control and Research Institute, Turkey), and Brucella IgM and IgG tests with the ELISA method (VIRCELL, Santa Fe, Granada, Spain). ELISA cut-off control values were accepted as >0.55 and <1.5 in accordance with the manufacturer's recommendations and considered the gold standard.
Agglutination in RBT and titers of 1/160 and above in the STA test were considered positive. Brucella ELISA IgG and IgM levels >11 (VIRCELL, Santa Fe, Granada, Spain) were considered positive. The study was approved by the Near East University Scientific Research Assessment Ethics Committee (decision number: 2017/466 dated: 24.02.2017).
Statistical Analysis
The results were evaluated by using the SPSS version 15.0 (Statistical Package for the Social Sciences, United States) software. The sensitivity and specificity of the RBT and STA were calculated using ELISA IgG and IgM positivity as reference/ golden standard results.
Results
Of the 27 patients with positive serologic test results, RBT was positive in 21 (6.5%), STA was positive in 15 (4,6%), and ELISA (IgG and/or IgM) was positive in 10 (3.1%) of the patients (Table 2, 3). Six RBT-negative patients (28%) had positive ELISA results.
Seventeen samples (80.9%) were RBT-positive but ELISAnegative. Tables 2 and 3 shows the distributions of serological test results in the serum samples analyzed in our study. There was no statistically significant correlation between presence of Brucella IgG and sex, age, or years of livestock farming experience (p<0.05). Titers did not differ between rural and urban residents.
When ELISA was considered to be the gold standard, the sensitivity and specificity of Rose Bengal and STA tests were calculated as 40%, 60% and 60%, 72%, respectively.
Discussion
Patients do not always present characteristic signs and symptoms, and the disease may manifest clinically in different forms (acute, subacute, chronic, and localized), thus making diagnosis challenging[10]. Since asymptomatic patients were included in our study group, none were Brucella IgM-positive. Ten participants tested positive for Brucella IgG, 60% of which were animal breeders. Transmission to humans occurs through consumption of non-pasteurized milk and dairy products, contact with infected animal tissues, and inhalation or mucosal inoculation (nose, eyes, mouth) of infected aerosols[7]. We think that direct contact with infected material, one of the primary routes of brucellosis transmission, proves the results of IgGpositive participants.
Today, STA is the most commonly used serologic test worldwide[8]. In our study, 21 serum samples with positive RBT results underwent STA testing and 15 had titers higher than 1:160. False negative results should also be taken into account in the STA test. False negativity can result from testing within the first week of infection (titers <1:160 in the early bacteriological period); the presence of blocking antibodies (chronic brucellosis); invisible/ masked agglutination at low dilutions due to excess antibody in patient serum (prozone phenomenon); Brucella canis infection; and agammaglobulinemia[9]. We believe that these factors might have affected the positivity rate in our study by resulting in false negatives, and that it may be related to differences among the participants in regional means of livelihood and employment in livestock production. Since our study group consisted of asymptomatic participants, seroconversion study was not performed. Our findings of low sensitivity and specificity in the STA compared to other studies is likely due to the fact that the STA test could not be repeated 2 weeks later.
Conclusion
Ethics
Ethics Committee Approval: The study was approved by the Near East University Scientific Research Assessment Ethics Committee (decision number: 2017/466 dated: 24.02.2017).
Informed Consent: Consent form was filled out by all participants.
Peer-review: Externally and internally peer-reviewed.
Authorship Contributions
Design: K.S., T.Ş., M.S., Data Collection or Processing: M.G., M.Ö., E.G., A.A., Analysis or Interpretation: K.S., M.G., M.Ö., Literature Search: M.G., E.G., M.Ö., Writing: K.S., E.G., M.G., M.Ö., A.A.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.
