In the present study, we intended to shed some light on the still unveiled prevalence scenario of human brucellosis in Portugal, by conducting a retrospective study on about 2700 samples received at the Portuguese NIH over a 7-year period. Overall, serological diagnostic identified 167 (7.2%) positive cases of human brucellosis, of which 61.7% were male and half of the cases were in the age groups between 26-65 years. Preview studies show that in industrialized countries the disease mainly affects men aged between 20 and 45 years, and suggests that the distribution by gender is connected to occupational factor. In fact the people who work with farm animals, especially with cattle, sheep, goats and pigs (e.g., farmers, farm laborers, animal attendants, stockmen, shepherds, sheep shearers, goatherds, pig keepers, veterinarians and inseminators) are at risk through direct contact with infected animals or through exposure to a heavily contaminated environment. Although we found an irregular pattern of distribution of brucellosis by age groups, the infection rates calculated by molecular techniques revealed that the age groups between 46 and 65 years old are among the ones with the highest rates. This falls within the range of the one described in the ECDC "Annual epidemiological report:Food-and waterborne diseases and zoonose in 2014", in the European Union (EU), reflecting a higher number of cases registered in the age group 45 to 64 years old. We also observed a low infection rate in children (5.4%), which is in agreement with data from the European Food Safety Authority, reporting that the vast majority (80%) of the European cases of brucellosis were adults over 25 years. This lower infection rate in children when compared with the one observed in adults likely relies on the low contact of children with the common infection sources, such as infected animals and animal products.
Although the molecular diagnostic was only applied to a subset of samples from 2010 to 2016, from the 259 analyzed samples, 43 (16.6%) were positive for B. melitensis. The higher infection rates obtained when using real time PCR when compared with immunological methods are likely due not only to a probable higher sensitivity of the former technique, but also because, according to our experience, PCR is usually requested when the clinician has a strong suspicion of brucellosis (e.g., patients revealing complications associated with the disease). The majority of the PCR positive cases belonged to biovar 3, pointing it as clearly the most common species/biovar involved in the human disease in Portugal. Like other countries, Portugal, applies specific regulations and measures to eradicate the disease, however, regardless of the huge efforts to eliminate it, brucellosis has continued to be an endemic disease where B.melitensis biovars 1 and 3 amd B. abortus biovars 1 and 3 are the prevailing animals species.. This is in agreement with the data available for Europe. In fact, species information was provided for 99 of the 332 confirmed cases reported in the EU and Norway between 2008-2012, where 83.8% were reported to be B. melitensis. Although all clinical cases in Portugal were caused by B. melitensis, other Brucella species pathogenic to humans have been identified in animals, namely B. abortus and B. suis. This underlines the importance to perform the early detection and identification at species' level of the Brucella strains obtained from clinical samples (human and animals), which is a critical information to prevent or control the occurrence of outbreaks. For this reason, molecular techniques, such as the real time PCR, particularly when applied to patients with compatible clinical symptoms and negative serological findings, are the most useful approach for laboratory diagnosis due to the rapid and precise identification of the Brucella sp. strain present in the clinical sample.
The geographical distribution of brucellosis is constantly changing, with the emergence and reemergence of new outbreaks around the world. Reflecting the social, cultural, and economic policies that describe a changing global society, this pathology has been reflecting this dynamics, making its control and eradication a constant challenge.
In conclusion, despite the control and prevention measures implemented by the national authorities, brucellosis remains a problem in Portugal, with impact in public health and in the economy. This study draws attention to the importance of integrating clinical and laboratory data of human cases in order to increase the efficacy of the response measures, essentially in case of outbreaks. Furthermore, our findings reinforce the need to maintain an active epidemiological surveillance, enabling the early detection of all cases of infection and underlie the need to have a good communication flow between the human and animal Health Ministries, according to the One Health concept, the only valid way to improve the assessment of the actual epidemiological situation of brucellosis and other zoonosis in Portugal.