Biosafety and Biosecurity: Past and Present

Transcription

1 Biosafety and Biosecurity: Past and Present 2009 APHL Annual Meeting May 5-8, 2009 Karen B. Byers, MS, RBP, CBSP American Biological Safety Association (ABSA)

2 Method: Use Case studies training tools, illustrative examples. National Laboratory Training Network training Column in Applied Biosafety Contributor to CDC-NIH Biosafety in Microbiology and Biomedical Laboratories ASM lab practice committee; CLSI committee Harding and Byers,2006. Epidemiology of Laboratory-acquired infections. In Biological Safety, Principles and Practices, 4 th Ed. ASM Press rd.ed.

3 Reports and Investigations of Laboratory-Acquired Infections (LAI) Guideposts for good biosafety practice. Should not be a source of embarassment--lais occur whenever and wherever the links of the chain of infection connect. Reporting events is a service to profession, and a contribution to prevention efforts.

4 Past and Present LAI Case Studies Surveys Routine isolations: Condition of centrifuge safety cups M. tuberculosis Brucella N. meningitidis Shigella E.coli 0157 Exposures from: Stock Cultures (E.coli 057, B. melitensis, S. agona) Proficiency Test Samples

5 Statistical Data Pike and Sulkin: survey sent to 4,000 labs of various types: approx. 50% response. 4,079 laboratory-acquired infections from 1935 to % were from clinical laboratories. Harding and Byers: literature survey of LAIs from : 1,141 LAI and 24 deaths. 46% were from clinical laboratories. Most frequently reported in the literature were: M. tuberculosis (199), Brucella(143; 4 septic abortions) Shigella (66), Salmonella(64, 2 fatalities); N. meningitidis (32 infections, 11 fatalities) Harding and Byers, Epidemiology of Laboratory-acquired infections. In Biological Safety, Principles and Practices, 4th Ed. ASM Press

6 Top ten LAI * 1, 141 laboratory-associated infections total in literature survey Mycobacterium tuberculosis 199 cases Arboviruses 192 Coxiella burnetti 177 Hantavirus 155 Brucella 143 Hepatitis B 82 Shigella spp. 66 Salmonella spp. 64 Hepatitis C 32 Neiserria meningitidis 31 Harding and Byers, 2006, IN Biological Safety, Principles and Practices. ASM Press.

7 Survey-88 labs, large(>200beds; 32 small) hospitals or academic institutions, 3 reference labs. Shigella: 15 Brucella 7 (prophylaxis offered, no infection) Salmonella 6 S. aureus 6 (5 MRSA) N. meningitidis 4 Coccioides 2 C. difficilie 1 EJ Baron and M. Miller, Diagnostic Microbiology and Infectious Dis(60)(3)

8 Centrifuge safety cups-use, use, condition YES! No! No containmentgaskets missing! Case study: Blood tube broke in centrifuge. No safety cups. Aerosols generated exposed a clinical laboratory worker in Bolivia to Machupo virus, the cause of Bolivian Hemorrhaghic fever. MMWR 43(50):

9 Goals for Working Safely with Mycobacterium tuberculosis in Clinical, Public Health, and Research Laboratories 1-Laboratory safety requirements Table 2-Measures for controlling the risk for laboratory acquired tuberculosis TABLE%202

10 M. tuberculosis Aerosol route of transmission Biosafety cabinet defective or improperly certified; laboratory ventilation inadequate. Necropsy, Aerosol can refrigerant used to freeze tissue. Commonly used lab equipment in mycobacterial lab Well-written, practical advice in: CDC, 1981; Shireman, Grist, Grist and Emslie, Templeton.

11 Biosafety Cabinet should be re- certified at least annually Case study: 3 positive PPD tests occur simultaneously in a clinical micro lab. Check X-rays taken, INH offered. Investigation: faulty biosafety cabinetcontinuous re-circulation; no exhaust. One refused post-exposure treatment; later diagnosed with endometrial tuberculosis; culture yielded M. tuberculosis. Arch. Pathol. Lab.Med. 116:

12 Brucella US: about 150 cases/year. One of most frequent LAI Aerosol transmission, low dose required for infection. From: sniffing plates, spilling blood-culture bottle, mucocutaneous exposure to spray, aerosol from broken tube in centrifuge routine work outside of biosafety cabinet. MMWR, 57(02)

13 2 Recent Brucella cases Nov microbiologists in two laboratories from 2 unrelated isolates. Risk assessment for aerosol exposure: Post-exposure prophylactic (PEP) offered to staff at high risk of exposure. 146 staff were exposed. MMWR 57(02) 39-42

14 Brucella Exposure LOW Present in the laboratory during workup and identification of a Brucella isolate From the time the culture is first isolated until all culture isolates are removed or destroyed from the laboratory. Not meeting criteria (1,2,3) for high-risk. MMWR 57(02)39-42

15 HIGH: Brucella exposure-risk Assessment 1. Having direct personal exposure to Brucella (sniffing bacterial cultures, direct skin contact, pipetting by mouth, inoculation, or spraying into the eyes, nose,or mouth.) 2. Performing work on the open bench (outside of BL3) with an open culture plate or being in close proximity to such work (across an open bench top or within 5 feet) 3. Presence in the laboratory during any procedure on a Brucella isolate that might result in generation of aerosolized organisms and inhalational exposure (e.g. vortexing or catalase testing) -. MMWR 57(02) 39-42

16 Brucella MMWR 57(02) 39-42

17 Misidentification of B. mallei At the time of transfer on May 4, hospital A identified small, bipolar, weakly-staining Gram-negative rods in cultures of the liver abscess fluid. On May 5, Gramnegative bacteria also were isolated from the blood cultures. An automated bacterial detection system at hospital A initially identified the bacteria as Pseudomonas fluorescens/putida. However, subsequent studies of the same isolate performed at hospital B and CDC, including motility studies, cellular fatty acid analyses, and 16S ribosome sequencing, identified the organism isolated from the liver abscess as B. mallei. MMWR 2000 / 49(24);532-5

18 Use of Biosafety Cabinet in Clinical Labs for manipulation of sterile site samples See CDC advice online at: Sejvar, J. J., D. Johnson, T. Popovic, J. M. Miller, F. Downes, P. R. Weyant, D. S. Stephens, B. A. Perkins, & N. E. Rosentein Assessing the Risk of Laboratory- Acquired Meningococcal Disease. Journal of Clinical Microbiology 43(9), Available at: cgi?tool=pubmed&pubmedid=

19 N. meningitidis (Sejvar, 2005) Isolated approx. 3,000x/year in the US. 2 reports in 2002: MMWR 51(07): CDC did survey of professional organizations. Conclusion: rare, but serious consequences. 16 cases cases of probable laboratory-acquired meningococcal disease occurring worldwide between 1985 and 2001 were identified, including six U.S. cases between 1996 and Nine cases (56%) were serogroup B; seven (44%) were serogroup C. Eight cases (50%) were fatal. All cases occurred among clinical microbiologists. (50% compared to community rate of 10%)

20 Immunization Immunization recommended decreases risk of A, C, Y and W-135 (not B). Experience is not a sufficient immunization: 1 fatality- first isolate in that lab in 4 years. 1 fatality-state lab worker who worked with approx. 4 isolates/month.

21 Cumulative N. meningitidis LAI 32 to date; only person who worked with organism on bench infected. (droplet, not aerosol..but it doesn t take much!) 2 used plastic shield. made suspension did catalase assay Used loop to transfer culture Host factors: 1 individual had a cold 1 was short (droplet generation close to breathing zone?). 31 from Harding and Byers, New Zealand case.

22 Biosafety Cabinet for culture of sterile sites: blood, CSF, inner ear. CDC NPHL image #8406, CDC Meningitis and Special Pathogens Branch

23 Published LAIs may be just the top of the mountain

24 Ex: 4 Shigella LAI staff at one hospital ill; cultures yield S.sonnei. patient isolate from 1 wk earlier Pulsed-field electrophoresis same. 1 had worked with; 2 had not. Handwashing sink faucet contaminated LAI-staff member helped to clean biosafety cabinet where cultures manipulated. EJ Baron and M. Miller, Diagnostic Microbiology and Infectious Dis(60)(3)

25 Every Staff member has to be engaged in the Safety Program Ex: Journal of Clinical Microbiology. html Micro lab in 719 bed hospital; 16 technologists and 3 students. 6 became ill with S. sonnei; identical antibiograms. Year before: footpedal sink removed. Student stuck gloved finger in well with heavy suspension during typing. Used handwashing sink, instead of prep sink, to discard culture contaminated faucets.

26 Reminder: laboratory-acquired infections with 0157 have occurred with no accidents or breaches in infectious practice dose is very small: 50 to 100 organisms.

27 Glove, handwashing policy advice Spina, N., Zansky, S., Dumas, N. & Kondracki, S. (2005). Laboratory- Associated Infections with Escherichia coli O157. Journal of Clinical Microbiology 43(6): Available online at

28 4 E.coli 0157 LAI-PPE, tasks 3 buttoned lab coats; 1 unbuttoned. 1 working during county fair outbreak: glove use intermittent, answered phone w/ & w/o gloves. vortex, handle plates, automated ID system. Latex agglutination test. 1 wore gloves, washed after each removal, did not use on phone or computer. Made suspension w/swab, vortexed. (..students in lab) 1 did not wear gloves; did subculture only, no vortexing. 2 other staff worked on proficiency sample, 1 did not remove for sink or computer; did not HW after removal. 1-wore gloves, washed after removal. latex agglutination, no vortexing. Spina, 2005

29 Small infectious dose Case study: 6-year old child visiting lab, shown open plate--touched it. mother immediately washed hands with lab soap. child hospitalized 3 days later-colitis, haemolytic uremic syndrome. Plate was E.coli Required dialysis for 2 weeks, multiple transfusions. 1 year later normal renal function, blood pressure, no proteinuria. Salerno et. al Journal of Pediatrics145:

30 Laboratory Acquired Infections: Case Study from a Clinical Laboratory Evaluation Mike Miller, Ph.D., (D)ABMM, Centers for Disease Control and Prevention, Atlanta, Ga. --Summary prepared by Martha Boehme be vigilant, especially when working with small Gram negative or Gram positive rods. Hazardous procedures include sniffing plates, subculturing and picking colonies, making slides, inoculating biochemicals, and anything that generates an aerosol by imparting energy to a suspension shaking, stirring, pouring, pipetting, centrifuging, etc. Creating aerosols puts everybody in the lab at risk! Recommendations include conducting thorough risk assessments.. establish standard operating procedures, and enforce guidelines: Use engineering controls - biological safety cabinets ( BSC ), automatic faucets, shrink seal suspicious plates, plastic face shields, incinerator burners, alcohol hand gels, etc. Mandate use of BSC when slow growth on blood agar, no growth on MacConkey, Gram negative dipoloccci from blood or CSF Do not trust automated instruments or kits with unusual or slow-growing isolates Vaccinate workers in high-risk situations Establish procedures with medical response plans for each agent

31 Case study: Lab trainee is ill Bloody diarrhea, haemolytic uraemic syndrome (HUS). E coli 0157 isolated from fecal specimen; unusual phage type--54. Strange none isolated in lab.???????????????????????????????????

32 So what happened? The lab had one 0157 QC strain with the same phage type. Cultures were transposed at some point the 0157 was being used for E.coli antibiotic sensitivity testing. Not recognized 0157 also fully sensitive. PHLS Communicable Disease Surveillance Center CDR Weekly, Vol 6:no.28

33 1991 Brucella outbreak Case Study: 8 out of 26 microbiologists were infected with Brucella melitensis. 5 positive blood cultures for B. melitensis, biotype 3. After 1 confirmed case, did serology on lab staff; 8 had evidence of serologic evidence of infection; 7 had clinical illnesses between May and September. No laboratory isolation for 3 years..????? So what happened? Journal of Clin Micro (2)

34 Extensive investigation. Nothing in lab notebook, but a frozen vial had a date 6 wks.before the outbreak. Brucella isolate from a patient hospitalized 3 years earlier had been thawed and subcultured without a biosafety cabinet out on the open bench. Aerosol exposure infected staff had all worked in lab during manipulation. Journal of Clin Micro :

35 Journal of Clin Micro (2)

36 Recommended stock culture storage-- Both Publications recommended storage of stocks on slants, in screw-capped tubes not plates. Previous audience comment: Recommend Safety Labeling on stock cultures USE BSC or BL3

37 Aftermath- 2 secondary infections 1991 Brucella outbreak First recorded case of sexual transmission of B. melitensis. Technician in CDC reference lab with 26 years of experience was infected when handling these isolates.

38 Repercussions outside lab: Misdiagnoses of Tuberculosis Resulting From Laboratory Cross-Contamination Contamination of M. tuberculosis Cultures CDC's National Tuberculosis Genotyping and Surveillance Network (NTGSN): DNA fingerprinting --11 isolates from previously reported TB cases among persons in New Jersey whose DNA fingerprints matched the avirulent laboratory M. tuberculosis control strain H37Ra. MMWR 49(19);

39 S.typhi and S. agona proficiency sample handling or student w/ unknown? A microbiology laboratory director provided a student with S. typhi and S. agona strains as unknowns to work on. Director did standard re-isolation and identification on stock strains from previous proficiency samples. Went home every day and made dinner for his wife and two children. Wife was hospitalized and blood cultures yielded both S. typhi and S. agona; fatal. Son was also hospitalized, blood cultures were positive for S. typhi, but he survived. Strains infecting wife and son identical to the laboratory stock strains. Student and director did not become ill.. (Blaser and Loftgren, 1981)

40 Proficiency Tests & LAI cases of S.typhi from proficiency test sample (Blaser and Feldman 1980); out of 24 total LAI w/ S. typhi. Blaser & Lofgren (added secondary) S. flexneri dysentery. Jacobsen et al 1985 Grist and Emslie-3 S.tyhpi in UK1985,1991; no longer sent as proficiency sample in UK.. Diptheria. Chin, 1998.

41 2007 LPS Exercise Category B pathogen sent to 1,316 labs in US and Canada. Sent w/ letter directing handling in class II biosafety cabinet using BL3 primary barriers and safety equipment. Shows the reporting system works! Mislabeled sample sent to NYSDOH, handled on bench, 17 exposed.

42 Aftermath Reported to CDC-Survey of NY labs.-16, or 80% of participants, handled on bench. CAP did survey; 254 laboratories with 916 exposed (74%) with high-risk and 237 (26%) with low-risk exposures. 69% of those exposed received prophylactic antibiotics; however, 19 (73%) reported at least 1 systemic symptom. 7 had persistent symptoms for 16 months.

43 Practice Highlights: Needed Biosafety Level 3 practices all specimen handling in biosafety cabinet. Recommended: American Society for Microbiology. Sentinel laboratory guidelines for suspected agents of bioterrorism: Brucella species. Washington, DC: American Society for Microbiology; adfilename/ /brucella pdf.

44 Update: Potential Exposures to Attenuated Vaccine Strain Brucella abortus RB51 During a Laboratory Proficiency Test --- United States and Canada, 2007 January 18, 2008 / 57(02); ml/mm5702a2.htm

45 US Case studies: Biosecurity

46 Malicious use of stock culture: free muffins in the break room! 12 of 45 lab members: severe diarrheal illness. 8 had S. dysenteriae isolated from stool 4 hospitalized. Uneaten muffin, stool isolates; identical by pulsed-field gel electrophoresis to lab stock strain a portion of which was missing!! Kolavic et.al. JAMA 258 5)396

47 Community outbreak of Salmonellosis 2 waves between Sept. and Oct. 751 persons with Salmonella gastoenteritis; all ate at local restaurant salad bars. Religious commune had a laboratory, S. typhimurium strain in their lab matched outbreak strain. Criminal investigation: intentional contamination. Torok et. al JAMA 278(5) 389

48 Amerithrax investigation 2001, when anthrax spores were sent through the mail, causing five deaths and 17 infections, since this trajic biosecurity lapse did not originate from authorized laboratory research

49 Are LAI inevitable? Risk can certainly be minimized we all have work to do. There s a lot we can do..

50 Safety Improvements --now standard, were once a change.. Anna Pabst, bacteriologist of the US Public Health Service, Died Dec.25 of meningitis. Miss Pabst received the infection Dec.17, When a squirming animal into which she was injecting the Meningitis culture caused misdirection of the serum into her eye. She continued working until Dec.21, when she became ill. She was 39 years of age. (JAMA, 1936). Luer-lok syringes would have prevented this incident! Mechanical pipetting devices no more mouth pipetting. Rigid Needle disposal containers Gloves Safety engineered needles Plastic or mylar-wrapped capillary tubes

51 Clinical laboratories bench bench or bsc? CDC advice: START in the biosafety cabinet with cultures from sterile sites Blood, cerebrospinal fluid, inner ear. J. Clin Micro. 43(9)

52 Lab-specific Biosafety manual Emphasize biosafety as an ongoing laboratory community effort. Executive management support statement on first page. Specific Procedures, procedures, procedures- include engineering controls to be used (biosafety cabinet? Fume hood? Safe use of equipment references available. Include BMBL, Agent Summary Statements, ASM website links for BT agents. Incident follow-up Management statement that reporting exposures provides proper medical attention in a timely manner. Non-punitive system for Exposure reporting. Explain that follow-up to incident is important to prevent re-occurrence. Spell out a non-compliance policy. [example safety training, practices tied to performance review. } Staff meeting- go over one aspect of safety manual. Improve understanding, compliance. example: glove removal Acknowledge community service in performing safety tasks thank staff who drain eyewashes, safety showers, call to get more paper towels, etc

53 Glove Policy When to wear Where to wear When to remove Handwashing after removal Technique for handwashing

54 Glove removal- step 1&2 Images from Univ. of Maryland EH&S website.

55 Steps 3&4&5

56 Steps 6& 7

57 To sum up: Laboratory-Acquired Infections 1)There is no national reporting system; no statistical epidemiological data. 2) Reports in the scientific literature tell us that laboratory-acquired infections can and do occur. Insight, but not statistics, are available from literature surveys. Clinical laboratories: 471 bacterial LAI between Fatalities: 11 N. meningitidis, two Salmonella (1 one was secondary) ; four fetuses were fatally infected by B. melitensis. Biological Safety Principles and Practices, 4 th edition.2006.fleming, Hunt Ed. ASM Press

58 CDC Biosafety Resources Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5 th Edition Primary Containment for Biohazards: Selection, Installation, and Use of Biological Safety cabinets.

59 ning/page1057.html CDC also offers online course in biosecurity CDC has free online training course on biosecurity at: Labs need to discuss, and write up procedures to address their site-specific issues.