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Inadvertent Laboratory Exposure to Bacillus anthracis --- California, 2004

On June 9, 2004, the California Department of Health Services (CDHS) was notified of possible inadvertent exposure to Bacillus anthracis spores at Children's Hospital Oakland Research Institute (CHORI), where workers were evaluating the immune response of mice to B. anthracis. This report summarizes the subsequent investigation by CDHS and CDC, including assessment of exposures, administration of postexposure chemoprophylaxis, and serologic testing of potentially exposed workers. The findings underscore the importance of using appropriate biosafety practices and performing adequate sterility testing when working with material believed to contain inactivated B. anthracis organisms.

On May 28, 2004, CHORI staff members injected 10 mice with a suspension believed to contain nonviable vegetative cells of B. anthracis Ames strain. The suspension was centrifuged and drawn into syringes on an open bench in the laboratory. The mice were injected in a separate animal-handling facility at CHORI. By May 30, all of the injected animals had unexpectedly died. The carcasses were removed from the cages, placed into a plastic biohazard bag, and frozen. The bedding was discarded as standard animal waste. The cages were sanitized in an automated washer.

On June 4, an additional 40 mice were injected with the same suspension. By June 7, all but one of these mice had died. All subsequent work was performed under a biological safety cabinet (BSC), and additional personal protective equipment (PPE) was used (e.g., protective clothing and gloves). Animal cages were brought into the BSC, and the surviving animal was euthanized. The carcasses were removed, placed into double biohazard bags, and frozen. The bedding and cages were autoclaved.

On June 8, a sample of the original suspension was cultured; one mouse that died after the second experiment was necropsied and samples for cultures were obtained from its liver and peritoneal cavity. Within 24 hours, these cultures grew nonhemolytic gram-positive rods. Colony morphology was consistent with B. anthracis.

Suspension material and cultures were transported to a California Laboratory Response Network (LRN) reference laboratory for further identification. The California LRN confirmed that the organisms isolated were B. anthracis by using polymerase chain reaction and gamma phage lysis assay. At CDC, antimicrobial susceptibility testing revealed that the isolates were susceptible to penicillin, ciprofloxacin, and doxycycline. Multiple-locus variable-number tandem repeat analysis confirmed that the isolates were genotype 62, consistent with B. anthracis Ames strain (1).

On June 9, CDHS personnel visited the laboratory and animal-handling facility at CHORI to review the incident and laboratory procedures. No spills, puncture wounds, animal bites, or scratches were identified; however, initial handling of the suspension included snapping lids of microtubes, ejection of pipette tips, and centrifuging. The centrifuge tubes had snap-down tops, and the rotor was covered with a gasket. The laboratory procedures might have potentially expelled small drops of suspension but were considered unlikely to have released infectious aerosols. Because staff members believed they were working with inactive organisms, they had performed these activities on an open bench, and appropriate PPE was not consistently used until after the deaths of the second group of mice.

As part of routine laboratory procedure, horizontal surfaces had been cleaned with a buffered bleach solution (1:10 dilution) at the end of each day. After laboratory workers recognized the possibility of exposure to viable B. anthracis spores, all laboratory surfaces and hoods were cleaned twice more with the bleach solution. The animal facility was also sanitized with bleach and a quaternary ammonium disinfectant.

Twelve persons were involved in either the laboratory or its animal-handling facilities. Three of these persons had direct contact with the bacterial suspensions, cultures, or infected animals. Although at low risk for inhalation of B. anthracis spores, to further reduce their risk, the three workers with direct contact were recommended for postexposure chemoprophylaxis for prevention of inhalational anthrax (i.e., either ciprofloxacin 500 mg or doxycycline 100mg, orally twice daily for 60 days) (2). The nine persons who worked in the laboratory or animal-handling facility but who did not have direct contact were offered the same chemoprophylaxis regimen. All 12 were additionally offered, but declined, anthrax vaccine under an Investigational New Drug (IND) protocol for postexposure prophylaxis (3).

Eight of the 12 potentially exposed persons opted to take chemoprophylaxis, including the three persons for whom the regimen was recommended. One person subsequently had a rash consistent with adverse reaction to ciprofloxacin; doxycycline was substituted. No other adverse effects from chemoprophylaxis were reported. None of the potentially exposed persons had symptoms consistent with anthrax.

Serum specimens collected from nine (75%) of the 12 exposed persons 3--6 weeks after exposure were negative for IgG antibodies to B. anthracis protective antigen (PA) by enzyme-linked immunosorbent assay (4). Three persons did not provide sera for evaluation, including one person who had direct exposure to the bacterial suspensions and cultures.

Further investigation revealed that the suspension had been prepared by a separate contract laboratory in March 2004 and contained an estimated 1.5 x 109 vegetative organisms per 1 mL of phosphate-buffered saline solution. After heating the suspension at 140ºF (60ºC) for 2 hours, the contractor reported that the suspension revealed no spores and had no growth after 48 hours of incubation on sheep blood agar.

A sealed, screw-top tube containing the suspension was shipped to CHORI in a double-compartment package on wet ice and arrived intact. The tube of suspension was stored in a refrigerator until used. The suspension had been prepared specifically for the research laboratory and was not distributed to other facilities. All contractor laboratory personnel had received anthrax vaccine, and the suspension was prepared under biosafety level 3 (BSL-3) conditions.

Leftover suspension from the incidents at the research laboratory were provided to CDC for quantification of viable organisms and to confirm the presence of B. anthracis spores. Sample dilutions were plated in duplicate on sheep blood agar. Approximately 2.0 x 106 colony-forming units (CFU) were enumerated per milliliter of suspension after 24 hours of incubation at 98.6ºF (37.0ºC). Comparisons of heat-shocked (149ºF [65ºC] for 30 minutes) and non--heat-shocked samples at CDC indicated that the suspension primarily contained B. anthracis spores.

Reported by: A Lucas, PhD, Children's Hospital Oakland Research Institute; M Doane, MD, J Rosenberg, MD, D Gilliss, MD, P Duffey, PhD, D Sesline, DVM, D Lindquist, MPH, R Das, MD, B Materna, PhD, D Vugia, MD, California Dept of Health Svcs. S Reagan, MPH, M Fischer, MD, N Marano, DVM, A Hoffmaster, PhD, V Semenova, PhD, S Martin, MT, C Quinn, PhD, Div Bacterial and Mycotic Diseases; J Patel, PhD, Div of Healthcare Quality Promotion, National Center for Infectious Diseases; M Kiefer, R Ehrenberg, National Institute for Occupational Safety and Health; R Weyant, PhD, Office of Health and Safety; B Ellis, PhD, T Jones, L Bane, M Hemphill, PhD, Office of Terrorism Preparedness and Emergency Response, Office of the Director, CDC.

Editorial Note:

The findings in this investigation indicate that workers in a research laboratory unknowingly received and used a suspension from a contract laboratory that likely contained viable B. anthracis organisms. Manipulation of the suspension at the research laboratory was determined unlikely to have expelled infectious aerosols, and exposed workers were considered at low risk for inhalation of spores. CDC continues to work with state agencies and other federal agencies to investigate processing procedures at the contractor facility to determine why the suspension contained viable B. anthracis organisms.

B. anthracis spores are highly resistant to the effects of heat and chemical disinfection (5). Although the heat-killing procedures used by the contractor might have been lethal to vegetative cells, the procedures were not lethal to spores. Modifying suspension preparations by increasing the temperature and duration of heat-killing procedures or using formalin will increase the probability that spores are inactivated (5,6).

Inactivated suspensions of B. anthracis should be cultured both at the preparing laboratory before shipment and at the research laboratory several days before use to ensure sterility. Sensitivity of sterility testing might be enhanced by increasing the inoculum size and incubation time, and by inoculating in multiple media, including both solid and broth media. Such procedures would increase the probability of detecting even a small number of viable B. anthracis spores. CHORI staff members did not perform sterility testing on the suspension received in March 2004.

Because inhalation of viable B. anthracis spores can result in fatal infection, CDC recommends that laboratory personnel who routinely perform activities with clinical materials and diagnostic quantities of infectious cultures implement BSL-2 practices (7). These practices include use of appropriate PPE (e.g., gloves, gowns, or laboratory coats) and a BSC for procedures with the potential to expel infectious aerosols (e.g., centrifuging or ejection of pipette tips). Face protection (e.g., goggles, face shield, or splatter guard) should be used against anticipated splashes or sprays when potentially infectious materials require handling outside of the BSC. In the incidents described in this report, because CHORI staff members believed they were working with nonviable organisms, they did not fully implement BSL-2 practices until after the deaths in the second group of mice.

Research laboratory workers should assume that all inactivated B. anthracis suspension materials are infectious until inactivation is adequately confirmed. BSL-2 procedures should be applied to all suspension manipulations performed before confirming sterility. After sterility is confirmed, laboratory personnel should continue to use BSL-2 procedures while performing activities with a high potential for expelling aerosolized spores.

The Advisory Committee on Immunization Practices recommends routine anthrax vaccination of persons who work with production quantities or concentrations of B. anthracis cultures or perform other activities with a high potential for producing infectious aerosols (8). Facilities performing such work should have appropriate biosafety precautions in place to prevent exposure to B. anthracis spores; however, anthrax vaccination can be an additional layer of protection in the event of an unrecognized breach in practices or equipment failure. Because of the small potential for inadvertent exposure to aerosolized B. anthracis spores before or after sterility testing, vaccination might also be considered for researchers who routinely work with inactivated B. anthracis suspensions.

In addition, laboratories working with inactivated B. anthracis organisms should develop and implement training activities and incident-response protocols to ensure appropriate actions are taken in the event of a potential exposure. These protocols should describe mechanisms for offering counseling and postexposure chemoprophylaxis and obtaining paired sera from potentially exposed persons. Training at animal research facilities should emphasize prompt communication between animal handlers and researchers if animals are unexpectedly found dead and any special handling procedures are needed for carcasses and bedding. Finally, institutional biosafety committees should routinely review protocols and procedures to ensure that appropriate safety precautions are always in place.

References

  1. Hoffmaster AR, Fitzgerald CC, Ribot E, Mayer LW, Popovic T. Molecular subtyping of Bacillus anthracis and the 2001 bioterrorism-associated anthrax outbreak, United States. Emerg Infect Dis 2002;8:1111--6.
  2. CDC. Update: investigation of bioterrorism-related anthrax and interim guidelines for exposure management and antimicrobial therapy, October 2001. MMWR 2001;50:909--19.
  3. CDC. Use of anthrax vaccine in response to terrorism: supplemental recommendations of the Advisory Committee on Immunization Practices. MMWR 2002;51:1024--6.
  4. Quinn CP, Semenova VA, Elie CM, et al. Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen. Emerg Infect Dis 2002;8:1103--10.
  5. Turnbull PCB, Kramer JM. Bacillus. In: Murray PM, Baron EJ, eds. Manual of clinical microbiology. 8th ed. Washington, DC: ASM Press; 2003:349--56.
  6. Spotts Whitney EA, Beatty ME, Taylor TH Jr, et al. Inactivation of Bacillus anthracis spores. Emerg Infect Dis 2003;9:623--7.
  7. CDC, National Institutes of Health. Biosafety in microbiological and biomedical laboratories. 4th ed. Atlanta, GA: US Department of Health and Human Services, CDC, National Institutes of Health; 1999.
  8. CDC. Use of anthrax vaccine in the United States: recommendations of the Advisory Committee on Immunization Practices. MMWR 2000;49(No. RR-15).



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