Biosafety in Microbiology laboratory

1. Biosafety in Microbiology laboratory Dr.T.V.Rao MD Dr.T.V.Rao MD

2. BIOSAFETY: Preventing lab-acquired infections • Bacteria • Viruses • Fungi • Human blood, unfixed tissue • Human cell lines • Recombinant DNA Dr.T.V.Rao MD

3. Why is Biosafety Important? • Laboratories recognize hazards of processing infectious agents • Guidelines developed to protect workers in microbiological and medical labs through engineering controls, management policies, work practices Dr.T.V.Rao MD

4. Standard Microbiological Practices • NOT permitted in laboratories: • Eating • Drinking • Smoking • Handling contact lenses • Pipetting by mouth • Storing food and drink Dr.T.V.Rao MD

5. Precautions so people researching or trying to identify organisms do not become infected While handling or testing clinical specimens, workers could accidentally infect themselves or coworkers Labs must adhere to very specific safety regulations to work with organisms that pose a threat to human health Biosafety Levels Dr.T.V.Rao MD

6. Laboratories divided on basis of nature of microbes • Labs divided into 4 biosafety levels; protective practices increase with each • Biosafety Level 1 labs - work with least dangerous agents, require fewest precautions • Biosafety Level 4 labs - have strictest methods because dealing with agents that are most dangerous to human health Dr.T.V.Rao MD

7. Barriers - Primary barriers • Primary barriers: physical barriers or personal protective equipment between lab worker and pathogen • Gloves, masks, special breathing apparatuses Dr.T.V.Rao MD

8. BarriersSecondary barriers: • Secondary barriers: structural aspects of the laboratory that make working environment safer against infection • Sinks for handwashing, special containment areas, special air ventilation patterns  Dr.T.V.Rao MD

9. Universal Precautions • Include hand hygiene, gloves, gown, masks, eye protection, face shields, safe injection practices • Require that all equipment or contaminated items are handled to prevent transmission of infectious agents • Special circumstances may require additional precautions • Protective clothing, special site decontamination Dr.T.V.Rao MD

10. Risk groups, biosafety levels, practices and equipment

11. CDC/NIH: Biosafety Level 1and Level 2. Dr.T.V.Rao MD

12. CDC/NIH: Biosafety Level 3and Level 4. Dr.T.V.Rao MD

13. Levels of Containment • BL1 - microorganisms that don’t consistently cause disease in healthy adults • E. coli K12, S. cerevisiae, polyomavirus • Basic laboratory • Standard Microbiological Practices Dr.T.V.Rao MD

14. Biosafety Level 1 (BSL-1) • Agents not known to cause disease in healthy adults • Some organisms may cause disease in immunocompromised individuals • Agents include Bacillus subtilis, Naegleria gruberi, infectious canine hepatitis virus, non-pathogenic E. coli species Dr.T.V.Rao MD

15. Standard practices required: frequent handwashing door that can be kept closed when working; limits on access to the lab space when working; no smoking, eating, drinking, storage of food in laboratory; care to minimize splashes and actions that may create aerosols (tiny droplets); decontamination of work surfaces after every use after any spills; Biosafety Level 1 (BSL-1) Dr.T.V.Rao MD

16. Biosafety Level 1 (BSL-1) • Standard practices (continued): • decontamination of laboratory wastes; • use of mechanical pipettes only (no mouth pipetting); • "sharps" precautions, including special containers for disposing of needles and other sharp objects; • maintenance of insect/rodent control program; • use of personal protective equipment (lab coats, latex gloves, eye protection or face shields) • Open bench top sink for hand washing Dr.T.V.Rao MD

17. Biosafety Level 2 (BSL-2) • Agents associated with human disease • Generally required for any human-derived blood, bodily fluids, tissues in which infectious agent may be unknown • Agents include measles virus, Salmonella species, pathogenic Toxoplasma, Clostridium botulinum, hepatitis B virus Dr.T.V.Rao MD

18. Levels of Containment • BL2 - microorganisms of moderate potential hazard, transmitted by contact, ingestion, puncture • Salmonella, herpesvirus, human blood • Basic laboratory • Standard Practices PLUS Dr.T.V.Rao MD

19. Primary hazards: accidental needle sticks exposure to eyes and nose (mucous membranes) ingestion of infectious materials Agents do not cause lethal infections, are not transmissible via airborne route (do not cause infection if tiny droplets become airborne and are inhaled, which might occur if the material were spattered) Agents are pathogens for which immunization or antibiotic treatment is available Extreme care should be taken with contaminated needles and sharp lab instruments Biosafety Level 2 (BSL-2) Dr.T.V.Rao MD

20. Risk Group 2 Pathogenic for humans Unlikely a serious hazard Treatment and preventive measures available Limited risk of spread of infection

21. Standard practices include BSL-1 plus: policies to restrict access to lab; biohazard warning signs posted outside lab; surveillance of laboratory personnel with appropriate immunizations offered; biosafety manual with definitions of needed waste decontamination or medical surveillance policies; supervisory staff who have experience working with infectious agents and specific training for laboratory personnel in handling these agents Biosafety Level 2 (BSL-2) Dr.T.V.Rao MD

22. Primary barriers: biosafety cabinets or other approved containment devices Personal protective equipment: lab coats, gloves, face protection as needed Protective clothing removed when personnel leave laboratory area Cabinets thoroughly decontaminated daily and monitored for radiation for personal protection Secondary barriers: BSL-1 barriers plus autoclave for glassware Biosafety Level 2 (BSL-2) Dr.T.V.Rao MD

23. Levels of Containment • BL3 - microorganisms that cause serious disease, transmitted by inhalation • M. tuberculosis, yellow fever virus, hantavirus, Y. pestis (plague) • Containment lab: double door entry; directional airflow; all work in biosafety cabinet Dr.T.V.Rao MD

24. Agents with potential for respiratory transmission, may cause serious and potentially lethal infection May be studied at BSL-2 for diagnosis  Biosafety Level 3 (BSL-3) • Agents include Mycobacterium tuberculosis,St. Louis encephalitis virus, Francisella tularensis, Coxiella burnetii () Dr.T.V.Rao MD

25. TB diagnostics and laboratory strengthening • Care of patients with tuberculosis starts with a quality assured diagnosis, obtained by growing and identifying Mycobacterium tuberculosis from clinical specimens and conducting DST of the organism to confirm or exclude resistance. Uptake of TB diagnostic technologies requires appropriate laboratory infrastructure and adequate policy reform at country level to enable their effective use in TB screening and diagnostic algorithms • Laboratory infrastructure, appropriate biosafety measures and maintenance Equipment validation and maintenance Specimen transport and referral mechanisms Management of laboratory commodities and supplies Laboratory information and data management systems Laboratory quality management system are a priority. Dr.T.V.Rao MD

26. Mdr – Tb , xmdr-tb and biosafety • With growing incidences of MDR-TB and XMDR-TB it is highly essential all Microbiology laboratories must install Grade 3 Biosafety cabinets to prevent exposure to Infection. It necessary precaution's are not taken a fraction of Medical and Technical personal will be infected with grave consequences. Dr.T.V.Rao MD

27. Risk Group 3 Pathogenic, cause serious disease Effective treatment and preventive measures usually available Little person-to-person spread Dr.T.V.Rao MD

28. Standard practices include BSL-2 plus: strictly controlled access to the lab; specific training for lab personnel in handling potentially lethal agents; decontaminating all waste; changing contaminated protective lab clothing, decontaminating lab clothing before laundering; institutional policies regarding specimen collection and storage from workers to establish exposure Biosafety Level 3 (BSL-3) Dr.T.V.Rao MD

29. Primary barriers: Similar to BSL-2 personal protective equipment Respiratory equipment if risk of infection through inhalation Secondary barriers: All BSL-2 barriers Corridors separated from direct access to lab Access through self-closing double doors Air handling systems to ensure negative air flow (air flows into the lab) Air pumped into lab not re-circulated in building Biosafety Level 3 (BSL-3) Dr.T.V.Rao MD

30. Levels of Containment • BL4 - microorganisms that cause lethal disease, with no known treatment or vaccine • Ebola virus, Marburg virus • Maximum containment lab; positive pressure ventilated suits (moon suits) Dr.T.V.Rao MD

31. Risk Group 4 Lethal, pathogenic agent Readily transmittable • direct, indirect Effective treatment and preventive measures not usually available

32. Dangerous and exotic agents with high risk of life-threatening disease, aerosol-transmitted Related agents with unknown risk of transmission Biosafety Level 4 (BSL-4) • Agents (all viruses) include Marburg virus, Ebola virus, viruses that cause Congo-Crimean hemorrhagic fever, Lassa fever Dr.T.V.Rao MD

33. Primary hazards: respiratory exposure to infectious aerosols mucous membrane exposure to infectious droplets accidental sticks with needles or other sharp objects contaminated with infectious material For example In late 1960s, 25 laboratory-acquired Marburg infections, including 5 deaths Workers studying infected monkeys from Uganda First documented naturally-occurring human case occurred in 1975 Biosafety Level 4 (BSL-4) Dr.T.V.Rao MD

34. Personnel must receive specialized training in handling extremely dangerous infectious agents, containment equipment and functions Access to lab is restricted: immunocompromised persons are never allowed to enter the lab  Standard practices include BSL-3 plus: strictly controlled access to the laboratory; changing clothing before entering and exiting lab (showering upon exiting recommended); decontaminating all material exiting facility Biosafety Level 4 (BSL-4) Dr.T.V.Rao MD

35. Primary barriers: Biosafety cabinets used at other biosafety levels Full-body, air-supplied, positive pressure personnel suit Secondary barriers: All physical barriers at BSL-3 isolated zone or a separate building; dedicated supply and exhaust, vacuum, decontamination systems; a recommended absence of windows (or sealed and resistant to breakage) Biosafety Level 4 (BSL-4) Dr.T.V.Rao MD

36. Activity spectrum of select detergents and disinfectants

37. BSL-1: high schools, community colleges, municipal drinking water treatment facilities BSL-2: local health departments, universities, state laboratories, private laboratories (hospitals, health care systems), industrial laboratories (clinical diagnostic companies) BSL-3: state health departments, universities, private companies, industry, federal government (NIH, CDC) BSL-4: only 15 facilities in the US 9 federal (CDC, NIH), 4 university (Georgia State University, University of Texas Medical Branch), 1 state, 1 private Renovations underway at several labs, new facilities proposed at additional sites Laboratory Locations Dr.T.V.Rao MD

38. Standard Microbiological Practices • NEVER • recap, bend, or break needles • discard needles or sharps into biological waste bags • discard needles into regular trash Dr.T.V.Rao MD

39. Laboratorians have long recognized hazards of processing infectious agents Biosafety guidelines developed to protect workers in microbiological and medical labs through a combination of safeguards including engineering controls, management policies and work practices. Issue described differences between biosafety levels Help you understand process labs may have to undertake to identify microorganism, why every lab cannot test for every organism Biosafety is everyone's concern Dr.T.V.Rao MD

40. References • UNC School of Public Health Laboratory Safety Levels • WHO guidelines on laboratory training for Field Epidemiologists Dr.T.V.Rao MD

41. Programme created by Dr.T.V.Rao MD for Medical ,Technical and Health care workers in the Developing World • Email • doctortvrao@gmail.com Dr.T.V.Rao MD