Introduction

Brucellosis is a zoonotic disease that is transmitted to humans by infected animals or contaminated animal products. It is caused by Brucella species. Brucella abortus, Brucella melitensis, and Brucella suis are frequently identified as the causative agents in humans[1, 2]. Brucella spp. can be easily aerosolized and the infective dose is quite low, in the 10-100 range[2, 3]. Brucella spp. are responsible for 2% of laboratory-acquired infections, and actions/events that pose a risk of transmission to workers include sniffing bacteriological cultures, direct contact with broken skin, mouth pipetting, and sprays/splashes into the eyes/ nose/mouth during inoculation[1-4]. Herein, we present two cases of laboratory-acquired brucellosis that occurred as a result of direct contact with or inhalation of blood culture isolates.

A 26-year-old female patient working in a microbiology laboratory was admitted to the Infectious Diseases outpatient clinic with complaints of fever, joint pain, and fatigue for the last three days. On initial examination, her body temperature was 38 °C, blood pressure was 120/80 mmHg, and heart rate was 88/min. No pathological findings were noted in her physical examination. Results of laboratory testing were leukocyte count: 5,230/mm3 (neutrophil, 60.7%), platelet count: 309,000/uL, aspartate aminotransferase (AST): 34 U/L, alanine aminotransferase (ALT): 45 U/L, urea: 19.3 mg/dL, creatine: 0.85 mg/dL, sedimentation rate: 8 mm/h, and C-reactive protein: 1.75 mg/dL (N<0.8). The patient reported that she had been working in her current position for six months and did not have adequate experience in bacteriology. She also stated that there had been significant Brucella spp. growth in blood cultures within the past month and that she conducted bacteriological examination on an open bench outside the biosafety cabinet by sniffing and without using personal protective equipment. Based on this history, she was tested for brucellosis. Other than laboratory exposure, the patient had no history of eating fresh cheese or working with livestock. Rose Bengal test was positive and Brucella Coombs gel test (ODAK Brucella Coombs gel test, Toprak Medikal, İstanbul) showed a titer of 1/640. Gram-negative coccobacilli growth in her blood culture was identified as a Brucella spp. by conventional techniques. She was diagnosed with laboratory-acquired brucellosis and was administered oral doxycycline 100 mg twice daily and rifampicin 600 mg once daily for six weeks. No recurrence was observed during posttreatment follow-up.

The second patient was a 35-year-old female patient who had worked in the microbiology laboratory for 10 years. She presented to the outpatient clinic after her colleague was diagnosed with laboratory-acquired brucellosis. She reported no complaints in her history other than joint pain and fatigue.

Her temperature was 36.5 °C, blood pressure was 110/70 mmHg, and heart rate was 78/min. Laboratory testing showed leukocyte count: 6,680/mm3 (neutrophil, 60%), platelet count: 161,000/ uL, AST: 31.7 U/L, ALT: 43 U/L, urea: 26.6 mg/dL, creatinine: 0.73 mg/dL, erythrocyte sedimentation rate: 13 mm/h, and C-reactive protein: 0.54 mg/dL. Rose Bengal test was positive and Brucella Coombs gel test (ODAK Brucella Coombs gel test, Toprak Medikal, İstanbul) resulted in a titer of 1/160. The patient did not report any history of eating fresh cheese or working with livestock. Blood cultures performed to support a diagnosis of brucellosis yielded Gram-negative coccobacilli. The isolate was identified as a Brucella spp. by conventional techniques. She was treated with oral doxycycline 100 mg twice a day and 600 mg rifampicin once daily for six weeks. No recurrence was observed during post-treatment follow-up.

Brucellosis, caused by Brucella spp., is transmitted to humans through direct contact with the blood/aborted fetus/uterine secretions/placenta of infected animals and consumption of raw/non-pasteurized infected milk/dairy products[5]. Brucella infections pose a low risk for the general community but cause high-risk in terms of transmission to laboratory workers. It is included in risk group 3 in the microorganism risk classification of the World Health Organization[6]. Reported infection attack rates in the laboratory setting are between 30-100%, depending on personnel location and bacterial load[7, 8]. Studies investigating the development of laboratory-acquired brucellosis in laboratory personnel have reported widely varying rates such as 5.8%, 11.9%, and 43%[3, 7, 9]. After two cases of laboratory-acquired brucellosis occurred in our hospital, the other 10 laboratory workers were subjected to Brucella screening with serological tests. Other than the two cases reported, no additional cases were detected (overall 16.6%).

Breakage of blood culture bottles or centrifuge tubes is responsible for 20% of laboratory-acquired infections. However, other factors and behaviors that increase the risk of bacterial transmission include lack of experience on the part of the technician identifying Brucella isolates, careless examination of unidentified samples sent for analysis, inappropriate laboratory practices such as sniffing cultures and using open benches that do not meet biosafety level 3 requirements while working with Brucella isolates, not using personal protective equipment such as gloves, goggles, and masks, and practicing mouth pipetting. We determined that no reported laboratory accident preceded these cases. However, the first patient"s history included factors such as significant Brucella growth in blood cultures within the past month, inadequate experience in morphologic bacterial identification, and performing microbial identification on an open bench by sniffing, without the use of appropriate personal protective equipment in the laboratory. Despite being more experienced than the first patient, the second patient admitted were also doing the same wrong practices. Although both patients had received the requisite training in laboratory protection measures, their failure to adopt correct behaviors resulted in their infection.

A 2008 report from the Centers for Disease Control and Prevention (CDC) included a risk stratification for laboratory personnel exposed to Brucella[3]. According to this classification, high-risk exposure encompasses any person who handles a Brucella isolate in a class-2 biosafety cabinet without biosafety level 3 measures, who is within a five-foot radius of an open bench on which these activities are carried out, and all persons present in the laboratory during aerosol-generating events (e.g., centrifuging without sealed carriers, spills or splashes, and tube breakage)[3]. Low-risk was defined as all persons who are further than five feet from someone handling a Brucella isolate, without exposure to the high-risk scenarios described above[3, 4]. There is minimal risk of transmission to laboratory personnel when Brucella isolates are handled with biosafety level 3 measures in a class-2 biosafety cabinet[3]. Traxler et al.[3] reported that of 1724 laboratory personnel, 153 were exposed to Brucella spp. (49% of which were high-risk) and that only 0.3% of them developed laboratory-acquired brucellosis. Fiori et al.[8] reported that in personnel follow-ups conducted after laboratory accidents occurring in an experimental microbiology laboratory in Italy, 12 laboratory personnel working in various parts of the laboratory were serologically diagnosed with brucellosis (31% attack rate). The patients presented here handled Brucella isolates on an open bench, outside a biosafety cabinet, and were therefore evaluated as having had "high-risk exposure" based on the CDC risk classification.

In conclusion, microbiology laboratories are high-risk environments for the transmission of microbes. The main cause of almost all laboratory exposures that result in infection seems to be the "human factor"[10]. Every laboratory must have clear, concise, and easily accessible written procedures for the use of personal protective equipment, disinfection of equipment and contaminated materials, collection and processing of samples, waste handling, and cleaning spills and splashes[6]. Many laboratory-acquired infections may be prevented with the appropriate use of protective measures[10]. The cases presented here emphasize the importance of personal protective equipment and biosafety cabinet use among laboratory personnel and the need to suspect laboratory-acquired infections when laboratory personnel present to outpatient clinics.

Ethics
Informed Consent: Consent form was filled out by all participants.

Peer-review: Externally and internally peer-reviewed.

Authorship Contributions
Surgical and Medical Practices: M.S.S., D.B., Concept: M.S.S., D.B., Design: M.S.S., A.H.S., Data Collection or Processing: M.S.S., D.B., Analysis or Interpretation: D.B., A.H.S., Literature Search: A.H.S., M.S.S., D.B., Writing: M.S.S., A.H.S.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.