Biosafety in Microbiology Laboratory

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Biosafety in Microbiology Laboratory

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Biosafety in Microbiology laboratory:

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

BIOSAFETY: Preventing lab-acquired infections:

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

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 Why is Biosafety Important? Dr.T.V.Rao MD 3

Standard Microbiological Practices :

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

Biosafety Levels:

Biosafety Levels 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 Dr.T.V.Rao MD 5

Laboratories divided on basis of nature of microbes:

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 6

Barriers - Primary barriers:

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

Barriers Secondary 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 Barriers Secondary barriers : Dr.T.V.Rao MD 8

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 Universal Precautions Dr.T.V.Rao MD 9

Risk groups, biosafety levels, practices and equipment:

Risk groups, biosafety levels, practices and equipment BSL Laboratory type Laboratory practices Safety equipment 1 Basic teaching, research Good microbiological techniques None Open bench work 2 Primary health services; diagnostic services, research Good microbiological techniques, protective clothing, biohazard sign Open bench PLUS biological safety cabinet for potential aerosols 3 Special diagnostic services, research As BSL 2 PLUS special clothing, controlled access, directional airflow Biological safety cabinet and/or other primary devices for all activities 4 Dangerous pathogen units As BSL 3 PLUS airlock entry, shower exit, special waste Class III biological safety cabinet, positive pressure suits, double ended autoclave (through the wall), filtered air

CDC/NIH: Biosafety Level 1 and Level 2.:

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

CDC/NIH: Biosafety Level 3 and Level 4.:

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

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 Levels of Containment Dr.T.V.Rao MD 13

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 Biosafety Level 1 (BSL-1) Dr.T.V.Rao MD 14

Biosafety Level 1 (BSL-1):

Biosafety Level 1 (BSL-1) 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 ; Dr.T.V.Rao MD 15

Biosafety Level 1 (BSL-1):

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 16

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 Biosafety Level 2 (BSL-2) Dr.T.V.Rao MD 17

Levels of Containment :

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

Biosafety Level 2 (BSL-2):

Biosafety Level 2 (BSL-2) 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 Dr.T.V.Rao MD 19

Risk Group 2:

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

Biosafety Level 2 (BSL-2):

Biosafety Level 2 (BSL-2) 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 Dr.T.V.Rao MD 21

Biosafety Level 2 (BSL-2):

Biosafety Level 2 (BSL-2) 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 Dr.T.V.Rao MD 22

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 Levels of Containment Dr.T.V.Rao MD 23

Biosafety Level 3 (BSL-3):

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

TB diagnostics and laboratory strengthening :

TB diagnostics and laboratory strengthening Dr.T.V.Rao MD 25 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.

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 . Mdr – Tb , xmdr-tb and biosafety Dr.T.V.Rao MD 26

Risk Group 3:

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

Biosafety Level 3 (BSL-3):

Biosafety Level 3 (BSL-3) 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 Dr.T.V.Rao MD 28

Biosafety Level 3 (BSL-3):

Biosafety Level 3 (BSL-3) 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 Dr.T.V.Rao MD 29

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) Levels of Containment Dr.T.V.Rao MD 30

Risk Group 4:

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

Biosafety Level 4 (BSL-4):

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

Biosafety Level 4 (BSL-4):

Biosafety Level 4 (BSL-4) 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 Dr.T.V.Rao MD 33

Biosafety Level 4 (BSL-4):

Biosafety Level 4 (BSL-4) 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 Dr.T.V.Rao MD 34

Biosafety Level 4 (BSL-4):

Biosafety Level 4 (BSL-4) 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) Dr.T.V.Rao MD 35

Activity spectrum of select detergents and disinfectants :

Activity spectrum of select detergents and disinfectants BG+ BG- MycoB Spores Yeast Virus Prions Alcohol 70° ++ ++ ++ 0 + + 0 Aldéhydes +++ +++ ++ + +++ ++ 0 Ammonium IV +++ + 0 0 + + 0 Anilides + 0 NP NP 0 NP 0 Chlorhexidine +++ ++ 0 0 + + 0 Cl compounds +++ +++ ++ ++ ++ ++ + (a) Iodine (+ der.) +++ +++ ++ ++ ++ ++ 0 Hg compounds ++ ++ 0 0 + 0 ou + 0 Phénols : Variable activity depending on components (b) Hexachlorophène +++ + 0 0 + 0 0 (a ) Bleach (6%) during 60 min at 20°C ; (b) discussion on efficacy of phénol on prions

Laboratory Locations:

Laboratory Locations 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 Dr.T.V.Rao MD 37

Standard Microbiological Practices :

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

Biosafety is everyone's concern:

Biosafety is everyone's concern 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 Dr.T.V.Rao MD 39

References :

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

PowerPoint Presentation:

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