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Microbiology laboratories: some relevant definitions and
elements of biosafety
Relevant definitions
Pathogens
• microorganisms or infectious agents (bacteria, fungi, viruses, rickettsiae, chlamydiae, mycoplasmas, parasites and prions) capable of causing disease in humans, animals or plants
Relevant definitions
Infection• Invasion by and multiplication of a microorganism or
infectious agent in a bodily part or tissue ± tissue injury and progress to disease
Colonisation
• first stage of infection; pathogen situated at possible appropriate entry site (urogenital, digestive, respiratory, conjunctiva) – not necessarily followed by infection
EBSA 2014 Ghent
Relevant definitions - continued
Infectivity
• Capacity of pathogen to enter, survive and multiply in a susceptible host
Pathogenicity • Capacity of pathogen to produce disease in a susceptible
host
Susceptible host
• Organism (human, animal) which can be infected by pathogen (defense mechanisms are defeated by pathogen)
EBSA 2014 Ghent
The infectious process
Relationship between PATHOGEN and HOST
• Dynamic: reciprocal influences• Specific:
– host & tissue specificity (certain pathogens selectively infect certain tissues/cells)
• Depends on:
– Virulence of pathogen vs. Susceptibility / Resistance of host
– Exposure (pathogen meets susceptible i.e. suitable host)
EBSA 2014 Ghent
The infectious process - continued
EBSA 2014 Ghent
New HOSTSusceptible?Age, Gender,
Genetics, Immunity, Prevention
EXITBody fluids /Containment
breach
RESERVOIRInfected host /
Lab sample
ENTRYRespiratory,Digestive,
Skin, Membranes
Some factors which influence the occurrence of infection
Pathogen related:•Survival time of pathogen outside (suitable) host•Infective dose (quantity of pathogen required to produce infection)•Transmission routes (inhalation, ingestion, percutaneous, sexual, etc)
Host related:•Susceptibility (age, immune status)•Preventive means (vaccination, others)
Survival outside host (after Walther B.A., Ewald P.W., 2004)
Variola virus • years - patient exudate, room temperature, dark
• ~ 80 days – vesicle fluid, room temperature, dark
Mycobacterium tuberculosis
• 90 – 300 days – sputum, room temperature, dark• 10 – 120 days – dust (floor/objects/fabric), room
temperature
Neisseria meningitidis
• 1 day – glass/plastic/fabric, suspension, room T°, dark˂Hepatitis B Virus
• - at least 7 days
EBSA 2014 Ghent
Survival outside host (continued)
Spores: reproductive structures adapted
for longtime survival in unfavourable
conditions
(etymology: ancient Greek spora = seed)
Bacterial spores - outer layer of keratin resistant to chemicals, staining and heat → bacterium able to stay dormant for years, protected from temperature differences, absence of air, water and nutrients
Spore forming bacteria: • Clostridium spp (e.g. Clostridium difficile, Clostridium tetani); • Bacillus spp (B. anthracis).
EBSA 2014 Ghent
Relevant definitions - continuedPathogen Minimal infective dose
S. pyogenes, S. aureus Under 103 CFU
Shigella (virulent strains) As low as 10 CFU
Bacillus anthracis 104 spores !!
Brucella species 10-100 organisms
Francisella tularensis 10-50 organisms
EBSA 2014 Ghent
EBSA pre-conference course, Basel, 18 June 2013
Classification of microorganisms Risk groups (1-4)
Risk group 1 (no / low individual & community risk)
• human / animal disease – unlikely
e.g. E.coli non-pathogenic strains, Lactobacillus spp.
Risk group 2 (moderate individual & low community risk)• ability to cause human / animal disease
• laboratory exposure → possible infection (severe)• availability of prevention & treatment
e.g. Corynebacterium diphtheriae, Haemophilus influenzae, E.coli, Orthomyxoviridae (Influenza viruses)
EBSA pre-conference course, Basel, 18 June 2013
Risk groups (1-4) (continued)
Risk group 3 (high individual & low community risk)
• serious human / animal disease• respiratory transmission (aerosolization)• no interpersonal respiratory transmission (usually) +
exceptions!*• availability of prevention & treatment
e.g. E.coli O157:H7, Francisella tularensis type A, Flaviviridae (West Nile Virus), Hepadnaviridae (HBV),
+ M. tuberculosis* (interpersonal respiratory transmission present)
EBSA pre-conference course, Basel, 18 June 2013
Risk groups (1-4) (continued)
Risk group 4 (high individual & high community risk)
• serious human & animal disease• respiratory transmission
• interpersonal transmission possible• NO prevention & treatment
e.g. Filoviridae (Ebola, Marburg), Arenaviridae (Lassa)
EBSA pre-conference course, Basel, 18 June 2013
Why risk groups?
• Requirements for sample processing
Premises (location, construction)Equipments
MaterialsStaff related requirements (number, qualifications, training)
Procedures
▼
Biosafety Levels (BSL)
EBSA pre-conference course, Basel, 18 June 2013
Biosafety Level I (BSL I)Agents Not known to cause diseases in healthy adults
Practices Standard microbiological practices
Primary barriers
None
PPE Laboratory coats, gloves, eye/face protection
Facility Lab bench and sink
EBSA pre-conference course, Basel, 18 June 2013
Biosafety Level II (BSL II)
Agents Known to cause diseases in healthy adultsTransmission: skin injuries, ingestion, mucous membrane exposure
Practices BSL I + limited access, biohazard sign, “sharps” precautions, Biosafety manual
Primary barriers
BSCs (biosafety cabinets) for splashes & aerosol producing procedures
PPE Laboratory coats, gloves, eye/face protection
Facility BSL I + autoclave available in facility
Biosafety cabinets (BSC)
EBSA pre-conference course, Basel, 18 June 2013
Biosafety Level III (BSL III)
Agents Indigenous / Exotic; serious / lethal diseaseTransmission by inhalatory exposure
Practices BSL II + controlled access, decontamination: all waste, all lab clothing
Primary barriers
BSCs for all manipulations
PPE Laboratory coats, gloves, eye/face protection +Respiratory protection
Facility BSL II + self-closing, double-door access; negative airflow into lab; entry airlock/anteroom
Respiratory protection
EBSA pre-conference course, Basel, 18 June 2013
Biosafety Level IV (BSL IV)
Agents Dangerous / exotic – high risk of aerosol transmission; frequently fatal infections; no vaccines / treatments
Practices BSL III + change clothing before entry & shower before exit; decontaminate all materials on exit
Primary barriers& PPE
Class III BSCs for all manipulations ORClass I / II BSCs + positive pressure suit
Facility BSL III + separate building; double-door autoclave, fumigation chamber, etc.
BSL4 laboratories – separate buildings, high security, limited access (only authorized persons)
BSL4 laboratories – bidirectional air filtration(HEPA filters retain particles ≤ 0.3μM)
BSL4 laboratories: changing room (all clothing + decontamination shower)
BSL4 – working area (double-door airlocks, waste decontamination by sterilization, vaccinated staff if vaccines
available, PPE: airtight pressurized suits, etc)
BSL4 laboratories: decontamination shower upon exit
EBSA pre-conference course, Basel, 18 June 2013
CORRELATION: Risk group – BSL
Risk group BSL Facility
1 I Teaching laboratories
2 II Public health, Diagnostic laboratories
3 III Special diagnostic, Reference, Research laboratories
4 IV Dangerous pathogens units
The above correlation is orientative – BSL is decided based upon RISK ASSESSMENT for each type of laboratory procedure