Closing the Brief Case: Misidentification of Brucella melitensis as Ochrobactrum anthropi by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS)

Husain Poonawala; Tracy Marrs Conner; David R. Peaper

doi:10.1128/JCM.00918-17

DOI: 10.1128/JCM.00918-17

ANSWERS TO SELF-ASSESSMENT QUESTIONS

For sentinel laboratories that are part of the Laboratory Response Network (LRN) without BSL-3 level work areas, which of the following methods is appropriate for the evaluation of a suspected Brucella isolate?
1. MALDI-TOF MS using specific research use only (RUO) or select agent databases
2. ASM-recommended basic biochemical tests in a class II BSC with standard PPE
3. A Vitek 2 Gram-negative identification panel with sample preparation in a BSC
4. ASM-recommended basic biochemical tests in a class II BSC with BSL-3 PPE

Answer: D. All sentinel laboratories should be prepared to follow rule out or refer pathways for select agents. These consist of basic biochemical tests that can exclude select agents. Testing of suspected Brucella isolates must be performed under BSL-3 conditions or in a class II BSC using BSL-3 PPE. If an organism is not ruled out, the specimen should be referred to an LRN reference laboratory for more definitive testing. No commercial test systems should be used to evaluate a possible select agent, regardless of its ability to correctly identify such an agent.

Which of the following select agents is expected to grow on MacConkey agar?
1. Brucella melitensis
2. Burkholderia pseudomallei
3. Francisella tularensis
4. Bacillus anthracis

Answer: B. Among the select agents, only Burkholderia pseudomallei and Yersinia pestis reliably grow on MacConkey agar (MAC). Burkholderia mallei has inconsistent or very delayed growth on MAC, while Brucella spp. and Francisella tularensis do not grow on MAC. The failure of a Gram-negative rod seen on Gram stain to grow on MAC should be concerning for a select agent. In contrast, Ochrobactrum anthropi demonstrates growth on MAC. Bacillus anthracis is a Gram-positive rod and should not grow on MAC.

The U.S. CDC differentiates between high- and low-risk Brucella exposures for laboratory personnel. Which of the following is routinely indicated for low-risk exposures?
1. Weekly symptom watch, with daily self fever check for 4 weeks
2. Postexposure prophylaxis with doxycycline and rifampin
3. Brucella species serology at 0, 6, 12, 18, and 24 weeks
4. Postexposure prophylaxis with a single dose of ciprofloxacin

Answer: B. The CDC stratifies exposures as high, low, and none. Based upon current CDC guidelines, individuals with both high- and low-risk exposures should perform weekly symptom checks and daily self fever checks for 24 weeks in addition to serological testing at 0, 6, 12, 18, and 24 weeks. The preferred postexposure prophylaxis for high-risk Brucella species exposures are doxycycline at 100 mg twice a day and rifampin at 600 mg four times a day for 3 weeks. Postexposure prophylaxis is not routinely indicated for low-risk exposures but may be indicated for immunosuppressed individuals after consultation with appropriate health care providers. There is no specific follow-up recommended for individuals with no risk. If symptoms develop, additional testing may be recommended. A single dose of ciprofloxacin is not appropriate postexposure prophylaxis for Brucella spp., regardless of risk; it is an appropriate regimen following exposure to Neisseria meningitidis.

TAKE-HOME POINTS

Infections with Brucella spp. are uncommon in the United States, with approximately 100 cases per year concentrated in a dozen states, but they are the leading cause of laboratory-acquired infections, representing a significant risk to personnel working in clinical microbiology laboratories.
Small Gram-negative rods/coccobacilli growing on blood and chocolate agar only should raise concern for select agents, including Brucella spp. Commercial identification systems (manual or automated, including MALDI-TOF MS) should not be used for the identification of potential select agents.
Manipulation of organisms with potential for being Brucella should occur in a BSC using BSL-3 personal protective equipment to rule out and refer according to ASM/APHL Sentinel Laboratory Guidelines. Manipulation under any other conditions may place staff at high risk of exposure.
Commercial test systems may misidentify Brucella spp., and suspicion for Brucella spp. should be raised when small, Gram-negative coccobacilli or rods are seen on a smear, with no growth on MacConkey agar.
There is no requirement that commercial test systems contain select agents in their databases, nor are they required to test select agents to verify potential misidentifications.
The CDC stratifies exposure risk, and postexposure prophylaxis, symptom self-monitoring, and sequential serological monitor are commonly performed after laboratory exposure.