Biosafety in Microbiology Laboratory

DR.T.V.RAO MD 1

Dr.T.V.Rao MD

BIOSAFETY IN

MICROBIOLOGY LABORATORY

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• Bacteria

• Viruses

• Fungi

• Human blood, unfixed tissue

• Human cell lines

• Recombinant DNA

BIOSAFETY: PREVENTING LAB-ACQUIRED INFECTIONS

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• 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?

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• NOT permitted in laboratories: Eating Drinking Smoking Handling contact lenses Pipetting by mouth Storing food and drink

STANDARD MICROBIOLOGICAL PRACTICES

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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

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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

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• Primary barriers: physical barriers or personal protective equipment between lab worker and pathogen• Gloves, masks,

special breathing apparatuses

BARRIERS - PRIMARY BARRIERS

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• 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:

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• 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

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 healthservices; 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 PLUSspecial 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

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CDC/NIH: BIOSAFETY LEVEL 1 AND LEVEL 2.

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CDC/NIH: BIOSAFETY LEVEL 3 AND LEVEL 4.

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• 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

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• 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)

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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;

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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

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• 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)

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• BL2 - microorganisms of moderate potential hazard, transmitted by contact, ingestion, puncture

• Salmonella, herpesvirus, human blood

• Basic laboratory

• Standard Practices PLUS

LEVELS OF CONTAINMENT

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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

RISK GROUP 2Pathogenic for humans

Unlikely a serious hazard

Treatment and preventive measures available

Limited risk of spread of infection

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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

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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

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• 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

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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

()

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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.

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• 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

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Pathogenic, cause serious disease

Effective treatment and preventive measures usually available

Little person-to-person spread

RISK GROUP 3

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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

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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

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• 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

RISK GROUP 4

Lethal, pathogenic agent

Readily transmittable• direct, indirect

Effective treatment and preventive measures not usually available

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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

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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

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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

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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)

ACTIVITY SPECTRUM OF SELECT

DETERGENTS AND DISINFECTANTS

BG+ BG-Myco

BSpores 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

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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

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• NEVER

• recap, bend, or break needles

• discard needles or sharps into biological waste bags

• discard needles into regular trash

STANDARD MICROBIOLOGICAL PRACTICES

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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

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REFERENCES

• UNC School of Public Health Laboratory Safety Levels

• WHO guidelines on laboratory training for Field Epidemiologists

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• Programme created by Dr.T.V.Rao MD for Medical ,Technical and Health care workers

in the Developing World• Email

• doctortvrao@gmail.com