in early november 2001 , a 57-year - old female laboratory worker ( laboratory worker 1 ) began experiencing nonspecific symptoms of malaise , vomiting , headache , lower leg cramping , anorexia , and fever . one week after onset of symptoms , she was evaluated for severe headaches at a local emergency room , where cerebrospinal fluid ( csf ) and blood cultures were collected . small , gram - positive bacilli were isolated and characterized as coryneform bacilli , which are usually interpreted as contaminants of unknown clinical importance . despite multiple hospital admissions , approximately 5 weeks after symptom onset , colleagues from the hospital microbiology laboratory where she was employed ( laboratory 1 ) drew her blood for culture again . after 5 days of incubation , gram - variable coccobacilli , the brucella serum agglutination test ( sat ) was reactive ( 1:640 ) at the nysdoh laboratory , wadsworth center . laboratory worker 1 was initially treated with doxycycline and gentamicin , followed by doxycycline and rifampin , for 6 weeks of outpatient therapy . the isolate was later identified as b. melitensis by the wadsworth center and confirmed by the centers for disease control and prevention . the patient has not relapsed 18 months after completing treatment . in a second incident in mid - january 2002 , a 48-year - old woman had nocturnal temperature spikes to 40c , chills , drenching sweats , and weight loss . symptoms persisted , and uveitis developed . in early march 2002 , a diffuse , erythematous rash appeared on the anterior aspect of both legs . a blood culture and serologic tests for lyme disease , ehrlichiosis , and rocky mountain spotted fever ( rmsf ) were performed . from the blood culture , gram - positive cocci were isolated and identified as micrococcus spp . by a commercial laboratory . rmsf titers were immunoglobulin ( ig ) m - negative with a reactive igg of 1:256 . subsequently , she was referred to an infectious disease specialist , who found repeat rmsf titers unchanged , which made acute rmsf unlikely . when interviewed by nysdoh staff , the patient reported that she was a laboratory worker ( laboratory worker 2 ) at laboratory 2 . her initial blood culture specimen , which had originally been identified as micrococcus , was reassessed by the commercial laboratory . the commercial laboratory referred the original isolate to the wadsworth center , where the isolate was identified as b. melitensis . . they denied traveling outside of the united states , consuming imported or domestic unpasteurized dairy products , knowing ill family or friends who may have traveled , attending events with potentially contaminated foods , or handling farm or laboratory animals . both laboratory workers denied any accidental contamination or spills in the laboratory during the 6 months before their respective illnesses . site visits to laboratories 1 and 2 were conducted by nysdoh . on the open bench at laboratory 1 a syringe was used to directly plate contents of the blood culture media bottle onto agar . subsequent biochemical tests , including a catalase test , which may generate aerosols by introducing hydrogen peroxide to the specimen , were performed on the open laboratory bench . at laboratory 2 , subculturing occurred in a similar manner , but it took place in a class ii biosafety cabinet . however , biochemical tests were performed on the open laboratory bench when gram stain results indicated that spores were not present . laboratory workers 1 and 2 wore gloves when processing specimens , and both denied having dermatitis or skin lesions on their hands . after the diagnosis of brucellosis in these two laboratory workers , serum samples from their co - workers from laboratories 1 and 2 were tested by brucella sat ; samples from seven of eight co - workers were nonreactive ( < 1:20 ) . a co - worker of laboratory worker 1 had an initial agglutination titer of 1:40 ( indeterminate ) and 1 month later had a repeat titer of < 1:20 ; she denied having symptoms . to determine the source of these laboratory workers ' infections , each laboratory reviewed gram stains of blood culture specimens processed within 3 to 6 months of symptom onset . laboratory worker 2 reviewed prior gram stain slides from laboratory 2 and identified one slide containing a questionable coryneform bacillus . this blood culture had been drawn from laboratory worker 1 during her visit to the emergency room of laboratory worker 2 's hospital , early in november 2001 . approximately 2 months before her illness , laboratory worker 2 had personally processed laboratory worker 1 's blood culture but had characterized the isolate as coryneform bacilli . wadsworth center staff reviewed the original gram stain from laboratory worker 1 's blood culture specimen and noted gram - variable organisms with similar size and shape to brucella organisms . however , laboratory worker 2 did not identify any other gram stains that resembled brucella organisms during the period before her illness . the likely source of laboratory worker 1 's infection was found after reassessing gram stains performed in laboratory 1 in august , september , and october 2001 . a 76-year - old woman visited the emergency room served by laboratory 1 in early september 2001 . the patient had had fever ( temperature 38.3c ) for 1 day and headache for 3 weeks . she received one dose of ciprofloxacin for a possible urinary tract infection and was discharged . after 4 days of incubation , a gram stain of this patient 's blood culture specimen showed tiny , gram - negative coccobacilli , reported as coryneform bacilli . as part of the investigation for the source of infection for laboratory worker 1 , this patient 's prepared gram stain slide was referred to the wadsworth center , where small , gram - variable cocci were identified . more than 1 year after being seen in the emergency room , this patient was offered brucella sat and a repeat blood culture . she refused brucella sat , and her physician did not identify symptoms consistent with brucellosis . six months after this identification , further experimental analysis by the wadsworth center with polymerase chain reaction tests performed on gram stain material detected b. melitensis dna ( wadsworth center , nysdoh , unpub . the patient was again contacted , and brucella sat ( > 1.5 years after her initial blood culture ) showed a titer of 1:80 ( indeterminate ) . , the patient denied any visits outside the united states since 1989 , when she emigrated from peru . although brucellosis is a rare disease in the united states , its potential use as a bioweapon highlights the need for accurate and rapid identification ( 15 ) . in this investigation , brucellosis was diagnosed weeks to years after initial positive blood cultures were misidentified , and laboratory personnel were unknowingly exposed to the organism . this investigation suggests that transmission occurred from the 76-year - old index patient to laboratory worker 1 , on the basis of b. melitensis dna found in the gram stain material . processing the index patient 's blood culture specimen on an open laboratory bench was most likely the reason for laboratory worker 1 's illness approximately 5 weeks later . the same mechanism of transmission probably occurred when laboratory worker 2 handled laboratory worker 1 's blood culture specimen on the open bench for biochemical testing . in these instances , blood culture bottle media were transferred to slides and agar without much risk for aerosolization , since contents were manipulated with a syringe . biochemical tests , however , included the catalase test , which creates bubbles as a result of exposure to 3% hydrogen peroxide in positive specimens . neither laboratory worker could identify any other possible sources of infection , and because brucellosis is a rare disease in new york , the connection between these three patients is plausible . with the initial interpretations of these gram stains as gram - variable , which resulted in misidentification of the organism by three different laboratories , nysdoh initiated an effort to educate clinical laboratories in new york state about the potential difficulties in characterizing the organism and the importance of primary gram stain interpretation . additional investigation into the staining properties of brucella spp . under various conditions is now in progress and may help differentiate brucella spp . from other organisms . because of immigration and foreign travel , brucellosis remains an occupational hazard for laboratory personnel , even in industrialized countries where animal control efforts have virtually eliminated the disease . because of the nonspecific symptoms and the rarity of the disease in the united states , healthcare providers may not consider brucellosis in a differential diagnosis . . moreover , improved communication among healthcare providers and laboratory personnel should facilitate prompt and accurate identification and appropriate handling of the organism . its potential use as a bioweapon necessitates that healthcare providers , as well as microbiologists in hospitals and commercial laboratories , be knowledgeable about the diagnosis , identification , and handling of brucella spp . reporting brucellosis cases to public health officials is another component in protecting others from this disease . public health officials should notify laboratory personnel who may have handled cultures taken from patients ultimately diagnosed with brucellosis . early notification of exposed personnel could lead to their timely diagnosis and treatment , should symptoms occur , and could prevent further laboratory exposures .
we report two laboratory - acquired brucella melitensis infections that were shown to be epidemiologically related . 
 blood culture isolates were initially misidentified because of variable gram stain results , which led to misdiagnoses and subsequent laboratory exposures . 
 notifying laboratory personnel who unknowingly processed cultures from brucellosis patients is an important preventive measure .