Occupational exposure to Brucella spp.: A systematic review and meta-analysis

Brucellosis is a neglected zoonotic disease of remarkable importance worldwide. The focus of this systematic review was to investigate occupational brucellosis and to identify the main infection risks for each group exposed to the pathogen. Seven databases were used to identify papers related to occupational brucellosis: CABI, Cochrane, Pubmed, Scielo, Science Direct, Scopus and Web of Science. The search resulted in 6123 studies, of which 63 were selected using the quality assessment tools guided from National Institutes of Health (NIH) and Case Report Guidelines (CARE). Five different job-related groups were considered greatly exposed to the disease: rural workers, abattoir workers, veterinarians and veterinary assistants, laboratory workers and hunters. The main risk factors and exposure sources involved in the occupational infection observed from the analysis of the articles were direct contact with animal fluids, failure to comply with the use of personal protective equipment, accidental exposure to live attenuated anti-brucellosis vaccines and non-compliance with biosafety standards. Brucella species frequently isolated from job-related infection were Brucella melitensis, Brucella abortus, Brucella suis and Brucella canis. In addition, a meta-analysis was performed using the case-control studies and demonstrated that animal breeders, laboratory workers and abattoir workers have 3.47 [95% confidence interval (CI); 1.47–8.19] times more chance to become infected with Brucella spp. than others individuals that have no contact with the possible sources of infection. This systematic review improved the understanding of the epidemiology of brucellosis as an occupational disease. Rural workers, abattoir workers, veterinarians, laboratory workers and hunters were the groups more exposed to occupational Brucella spp. infection. Moreover, it was observed that the lack of knowledge about brucellosis among frequently exposed professionals, in addition to some behaviors, such as negligence in the use of individual and collective protective measures, increases the probability of infection.

Answer: The importance of this occupational group in European countries (where brucellosis has been eradicated in some countries), as well as the mis diagnosis by physicians and the misidentification of the organism were discussed in the following sentence: "Brucella spp.
cultures must only be handled in laboratories with biosafety level 3 or higher (92); however, due the lack of specificity of the clinical signs caused by the disease, associated with the effectiveness of public policies in some European countries (19,31), where brucellosis occurs primarily among travelers, many physicians rarely raise the hypothesis of brucellosis when sending biological samples for laboratory analysis, leading to exposure to the agent during manipulation of the clinical material by the microbiologist (93). Misidentification of the organism also happens and puts the health of individuals who manipulate cultures at risk (63,67)."

Consider including S4 Appendix in the manuscript.
Answer: We inserted the S4 appendix in the manuscript as figure 1, as requested.
Although the systematic review is proficiently executed, the information is to a large extent not new. Therefore, the authors should discuss in dept which new information has been generated through their work. In this perspective, the meta-analysis is perhaps the most interesting part of this research.
Answer: The authors recognize that the innovations achieved with systematic review and metaanalysis have not been properly valued and discussed. We appreciate the suggestion and to overcome this deficiency we added the following sentences highlighting the new information and scientific contribution provided by the present study: "The lack of accurate information on the quantification and peculiarities of the risk of brucellosis in each occupational group makes it difficult to direct public resources for the control and prevention of brucellosis in individuals most likely to present the disease, especially in a context with several other demands which also require a portion of the available funds, already limited. In this context, this systematic review provided a meticulous understanding of the risk factors peculiar to each of the main occupations (farmers, slaughterhouses, veterinarians, laboratories and hunters) closely related to Brucella spp. infection. Our results also revealed the great lack of information from these occupational groups on the importance of applying preventive measures to minimize the risk of transmission of brucellosis during work. In addition, through meta-analysis it was possible not only to confirm the occupational character of brucellosis, widely recognized, but also to quantify this risk in an unprecedented way in the scientific literature through the calculation of odds ratios, systematically compiling studies so far dispersed in the literature."

Specific comments:
Title: not Ok, I suggest: Occupational exposure to Brucella spp.: a systematic review and metaanalysis Answer: We modified the tittle as suggested: "Occupational exposure to Brucella spp.: a systematic review and meta-analysis".

L27: live attenuated anti-brucellosis vaccines
Answer: We corrected the term as suggested.

L41: different wildlife species
Answer: We corrected the term as recommended.

L45: live attenuated anti-brucellosis vacines
Answer: We corrected the term as suggested.

L69-72: introduce the DALY concept
Answer: The DALY concept was inserted and exemplified with a study that performs its calculation for human brucellosis in India in 2018, both for occupational groups and for the adult population not occupationally exposed to Brucella spp.: "Damage caused by the disease in individuals' quality of life is intangible and the economic losses attributed to the infection in humans are associated to the costs of hospital treatment, drugs and absence from work due to disabling feature of the disease in its severe form (6). These damages are more intense in groups frequently exposed to microorganisms of the  (10), in which one DALY can be thought of as one lost year of "healthy" life."

L76: live attenuated anti-brucellosis vaccines
Answer: We corrected the term as suggested.

L167-169: specify to which animal species the aborted fetus belongs
Answer: We specify that abortion materials were from production animals: "Moreover, another study also identified that aborted fetus remains from production animals were abandoned in the pasture and eventually ingested by dogs and pigs, in some properties in Angola (33)."

L189: live attenuated anti-brucellosis vaccines
Answer: We corrected the term as suggested. the higher incidence of the disease in its territory (262.2 annual cases per million of population) has a smaller number of high quality scientific publications (three) (Pappas et al., 2006). Thus, the number of studies and cases of human brucellosis is much more related to the demand and interest of researchers on this subject than the impact that this disease has on each nation.  (Fig 3a)."

L343: all systematic review should follow the PRISMA statement
Answer: The authors thank the statement, however we kept this sentence in the study as the information that the PRISMA methodology is a strength and we add the comment recognizing that this methodology is recommended in all systematic review studies: "The greatest strengths of this paper are that it is based on the PRISMA statement (as recommended for conducting systematic reviews)".

L281: reference
Answer: We added the reference as recommended. "The close contact of rural workers with small ruminants, preferred hosts for B. melitensis, was identified as the main form of acquisition of the disease among these individuals (Table 1), which has been confirmed by the identification of a high genetic similarity between B. melitensis strains isolated from occupationally infected workers and from goat milk samples (38)."

L304: laboratory worker: see general comment
Answer: We corrected as suggested.

L325: hunters: for which countries is this group important?
Answer: The authors pointed out that such groups were found in two countries where hunting is cultural: "The occupation with the lowest number of infected individuals identified was the group of hunters (n = 10), which differently from the previous groups exhibited exclusively B.
suis isolates (n = 9) (Fig 3a). Hunting, a widespread activity in United States of America and in some European countries, such as France, is often associated with the primary route of Answer: We replace "expressive" for "considerable": "However, it is important to take into account that despite the low number of studies used in the meta-analysis, the total number of individuals analyzed (n = 1069) and those with occupational brucellosis (n = 269) was considerable, supporting the robust results observed (Fig 4)." L350: L354: The One Health concept comes out of the blue and should either be deleted or better explained Answer: The One Health concept was explained and contextualized in the following paragraph: "These data on human cases of occupational brucellosis can be used as a first step towards adopting a One Health approach, which is an interdisciplinary collaboration that aims to reduce the occurrence of zoonotic diseases in humans, through the prevention of such diseases in animals (95). Thus, the control of brucellosis could be conducted more efficiently and strategically, in order to reduce the incidence of the disease not only in humans, but also in animals and the environment."

Strengths:
The Answer: The authors acknowledge the comments about the manuscript.

Weaknesses:
The manuscript provided is a review or mini-review, but is laid out in a form reminiscent of a primary publication with appropriate sections (Introduction, Methods, Results, Discussion and Conclusion Answer: Although conventional reviews present the text continuously, divided into sections related to the subject, according to the PRISMA methodology, a meta-analysis should contain all sections of a standard article. Thus, in the results section, the information that returned in the systematic search was presented, while the discussion section is based on comments on the meaning of the results, the comparison with other research findings and the author's position on the subject. The entire text was strategically organized in order to first present the results of each occupational group from the most exposed to the least exposed, as well as the discussion, in order to avoid going back and forth in the same information repeatedly.

The bottom line is that the meta-analysis, which is a novel feature of the manuscript, is only briefly described. Overall the study provides little added insight relative to the human/animal cycle of infection, including only 6 lines of results in a manuscript exceeding 360 lines of text.
The results are by no means a surprise, since the level of occupational exposure/infection parallel the previous literature with or without a meta-analysis. The finding of a 3.5-fold increase in potential for acquiring disease for workers exposed to the agent over workers who are not potentially exposed provides little real-world usefulness other than to predict an increased likelihood that flu-like symptoms are more likely to be brucellosis related. However, the authors might consider if such an approach could be used to improve training.

Answer:
The authors appreciate the comment. Despite being a very relevant information for the study, the exposure of the results of the meta-analysis is quite simple, since it is a calculation of an odds ratio. However, this was not the only result related to the cycle of human and animal brucellosis infection. In this systematic review it was possible to identify each of the most recurrent risk factors for each occupational group most exposed: although they are all grouped as "most likely to come into contact with the pathogen", each of them has its particularities, such as the manipulation of uterine secretions from infected animals, neglects the use of personal protective equipment, accidental exposure to live attenuated vaccines, errors in the identification and manipulation of microorganism cultures and habits of eviscerating carcasses in environments with unfavorable conditions. Thus, the great relevance of the present study is not only for compiling such information, but also for quantifying the risk that these individuals are more likely to develop the disease when compared to individuals not exposed to work. The authors are also grateful because this reviewer's comment caused a perception that the importance of the work described above may not have been properly explored in the manuscript. Thus, in order to provide a better understanding and practical application of this study for readers, we added the following paragraphs related to the discussion of meta-analysis and the relevance of the study not only for the scientific environment but for its benefit to the community: "The occupational character of human brucellosis is supported by the results generated from the meta-analysis of 3 case-control studies, which showed that animal breeders, laboratory workers and abattoir workers were significant more likely to become infected with Brucella spp. strains than people who develop other job-related activities (OR 3.47; 95% CI: 1.47 to 8.18) (Fig 4). The low number of selected studies with a case control design (n = 3) observed among the articles resulted in the small number of high-quality papers eligible for meta-analysis. It occurred because of the impossibility of access to data of exposed and nonexposed individuals. However, it is important to take into account that despite the low number of studies used in the meta-analysis, the total number of individuals analyzed (n = 1069) and those with occupational brucellosis (n = 269) was considerable, supporting the robust results observed (Fig 4). Those data revealed the weight of exposure to Brucella spp. during labor activities for the occurrence of human brucellosis, which is essential to take into account for the design of strategies to minimize its occurrence. (…) The lack of accurate information on the quantification and peculiarities of the risk of brucellosis in each occupational group makes it difficult to direct public resources for the control and prevention of brucellosis in individuals most likely to present the disease, especially in a context with several other demands which also require a portion of the available funds, already limited. In this context, this systematic review provided a meticulous understanding of the risk factors peculiar to each of the main occupations (farmers, slaughterhouses, veterinarians, laboratories and hunters), closely related to Brucella spp. infection. Our results also revealed the great lack of information from these occupational groups on the importance of applying preventive measures to minimize the risk of transmission of brucellosis during work. In addition, through meta-analysis it was possible not only to confirm the occupational character of brucellosis, widely recognized, but also to quantify this risk in an unprecedented way in the scientific literature through the calculation of odds ratios, systematically compiling studies hitherto dispersed in the literature. Since the human cases of occupational brucellosis in this study present direct and continuous contact with animals or their fluids, responsible for the transmission of the pathogen and the development of the disease in these individuals, these data can be used as a first step towards adopting a One Health approach, which is an interdisciplinary collaboration that aims to reduce the occurrence of zoonotic diseases in humans, through the prevention of such diseases in animals (94). Thus, the control of brucellosis, which could be conducted more efficiently and strategically, in order to reduce the incidence of the disease not only in humans, but also in animals and the environment."

Specific Comments:
(lines 53-4) The report of annual incidence dates from 2006. The authors might consider the use of the approach documented to estimate or update the frequency of infection.
Answer: Unfortunately, after a wide search, as far as the authors are aware, this reference is still the most current with regard to the occurrence of human brucellosis worldwide.
The figures need to be separated from the text and provide greater detail so that they are understood without reference to the text.