PRION RESEARCH GUIDELINES
Creutzfeldt-Jacob Disease (CJD) is
one of a group of diseases called transmissible
spongiform encephalopathies, that also includes kuru and
Gerstmann-Sträussler-Scheinker syndrome of humans, scrapie of sheep and “mad
cow disease” of cattle. Long (months to
years) incubation periods precede clinical illness of chronic progressive
pathology that may last weeks to months.
CJD is characterized by progressive dementia, myoclonic fasciculations,
ataxia and somnolence. The clinical
course of CJD usually lasts several months and is invariably fatal. No effective treatment is available and
there are no known cases of remissions or recoveries.
Transmissible spongiform
encephalopathies are caused by a unique infectious agent called a prion (proteinaceous infectious particle),
composed of a proteinaceous material devoid of detectable amounts of nucleic
acid. These are abnormal versions of
prion protein, or “PrP” which is ubiquitous to cell membranes, but is highly
species specific. These particles do
not infect cells or tissues and propagate, but rather are able to convert
normal prion proteins into the abnormal form.
The conversion rate is logarithmic but slow, and disease is thought to
be from autoimmune attack against the abnormal membrane protein. . Much more common than transmitted prion
diseases are those associated with a genetic defect, and a significant
percentage of prion-caused disease is “spontaneous”, where there is no identifiable
reason for the patient’s protein to become abnormal.
Prions are unusually resistant to
standard means of inactivation, including formaldehyde (including fixing
tissues for histological study), ethanol and UV radiation. However, they can be inactivated by fresh
household bleach, 1.0M sodium hydroxide (NaOH), 4.0M guanidine reagents, phenol
and autoclaving. Procedures involving
brain tissue from patients with neurological degenerative disorders (such as
CJD and Alzheimer's disease) pose special challenges in reducing potential
exposure to prions; such material should be handled with at least the same
precautions as HIV+ or HBV+ human tissue.
Those working with non-human prion-infected materials must follow the
same precautions.
The CDC classifies prions as Risk
Group 2 agents requiring Biosafety Level 2 (BSL 2) containment. The UCSD Biosafety Committee (BSC) has
determined that work involving human or bovine central nervous system tissues
or primary cell lines created locally from these tissues, requires Biosafety
Level 2 (BSL-2) practices and procedures plus additional precautions which are
detailed below All researchers working
with these agents are required by the University of California, San Diego
(UCSD) to have a valid Biohazardous Materials Use Authorization (BUA);
Institutional Biosafety Committee approval must be granted before any research
can be initiated.
It is important that all
lab personnel (even those not directly working with the virus) be informed and
aware that prion research is being conducted in the lab. If your lab does not
have a copy of the Biosafety Manual it is available on the Web.
· All fixed,
non-fixed, or frozen tissues must be contained within watertight containers and
labeled with the universal biohazard symbol and the notation "Infectious
Materials".
· Signs and
labels must be placed to indicate each area where prions are used or stored
(including biosafety cabinets, incubators, refrigerators, laboratory entrance
doors, etc.)
· All vacuum
lines must be fitted with a HEPA filter (an example is the "Vacushield
™" in-line hydrophobic filter, Product # 4402 from Gelman Science).
· No work
with prion-infected tissues is permitted on the open bench. A Biosafety Cabinet
or other enclosure must be used for all manipulations including (but not
limited to):
a.
pipetting
b.
harvesting infected cells
c.
loading and
opening containers
d.
cutting, sectioning or dissecting tissues
· Centrifugation
must be done in closed containers and using sealed rotors.
· All
tissues, infectious waste and instruments (e.g., specimen containers, knives,
blades, cutting boards, and centrifuge tubes) used in the processing of such
samples must be decontaminated as described below.
· Personnel
must wear gloves and gowns while handling tissues which are potentially
contaminated. All protective clothing
must be removed before leaving the laboratory.
· Sonication
or homogenization of tissues must be performed in a properly certified Class II
biosafety cabinet.
· Microtome
blades and knives used for cutting tissue must be cleaned with an instrument
that does not put the hand or finger of the operator in or near contact with
the blade.
· Contaminated
liquids are disinfected by making 1.0N sodium hydroxide (NaOH) followed by
autoclaving at 132º C for 4.5 hours.
Alternatively (if an autoclave that can reach that temperature isn’t
available), the 1.0N NaOH-treated liquid can be held at room temperature for 24
hours. Liquids treated in either of
these ways can then be poured down the sink.
· Contaminated
surfaces that can withstand the treatment are cleaned with 1.0N NaOH, allowing
5 minutes of contact time, followed by wipe down with 1.0N HCl, then thorough
washing with clear water.
· Contaminated
surfaces that cannot withstand NaOH/HCl treatment are cleaned with 10%
household bleach, allowing 10-15 minutes contact time, then washed with clear
water.
· Contaminated
skin surfaces are washed with 1.0N NaOH or 10% bleach for 2-3 minutes, followed
by rinsing with copious amounts of water.
Splashes to the eye are rinsed with copious amounts of water or saline.
· Contaminated
dry waste is autoclaved at 132º C for 4.5 hours, then discarded as solid waste
(trash).
· Sharps
waste is autoclaved at 132º C for 4.5 hours before being picked up as medical
waste for incineration. Note: Autoclaving sharps is acceptable in this
case because of the elevated risks associated with prion exposure.
· Gloves
· Wrap
around outer clothing when introducing vector into animals or performing
necropsies. Lab coats are adequate for tissue culture manipulations.
· Goggles
(not to be confused with safety glasses)
· Remember
that symptoms of disease will not appear for months or years.
· Any skin
contact with possibly infectious materials should be followed by washing with
1N sodium hydroxide for two to three minutes, followed by extensive washing
with water.
· If someone
is exposed to prion-containing material by splash or skin puncture, or material
containing prions is spilled, after ensuring that the exposed person is
disinfected, the Principal Investigator (PI) must notify EH&S. The PI must also submit a written report to
the BSO regarding spills and accidents which result in overt exposure to
tissues. The report must include the following:
a) Specification
of amount released, time involved, and explanation of procedures used to
determine the amount involved.
b) Description
of the area involved and the extent of employee exposure.
c) Report of
medical treatment provided.
d) Corrective
action taken to prevent the reoccurrence of the incident.
e)
Prion decontamination procedures
RECORDKEEPING
·
Records must be maintained for a period of 30 years by
the PI.
·
If the employee is no longer employed by the University, or
if the employee retires or dies, or if the PI leaves the University and the
employee does not go with the PI, their records or notarized true copies must
be forwarded to the Director of National Institute of Occupational Safety and
Health (NIOSH).
·
Records must be provided upon request by representatives of
the Chief and/or Director of NIOSH.
·
Any physician who conducts a medical examination must
furnish the employer a statement of the employee's suitability for employment.
·
Access to the laboratory must be restricted to trained personnel
when work is being conducted on tissue that potentially contains abnormal
prions. Training must be documented,
and personnel handling tissues must be trained in the following:
a) Nature of
CJD
b) Route of
transmission of CJD
c) Specific
hazards associated with handling of the tissue(s).
·
Work with abnormal prions in animals requires prior approval
of the Animal Subjects Committee, and the Institutional Biosafety Committee.
·
Arrangements must be made with the laboratory animal care
office and the supervising veterinarian.
Special housing and precautions are required that will take extra effort
and expense to set up.
·
Special training must be given to all animal husbandry
personnel on prions, the hazards associated with the work, required practices
and procedures and proper handling of bedding, cage washing, and all other
husbandry materials associated with the experiment.