Parasites

Mycobacteria

Bacterial Spores

Both Cryptosporidium spp. oocysts and Giardia spp.
cysts are used as test organisms to demonstrate
germicidal effectiveness. Cryptosporidium has been
demonstrated to have a higher chemical resistance
and Cryptosporidium spp. oocysts are more readily
available than Giardia spp. cysts. Both are
significantly pathogenic (both have an infectious
dose of 10 cysts) and care is advised when “using
these microorganisms as parasitic biological
indicators.

Mycobacterium phlei has a demonstrated measure of
disinfectant resistance, is a rapid grower and is
pigmented for easy identification. M. bovis (BCG)
is used in the AOAC Tuberculocidal Method and is
analogous to M. tuberculosis in that it is in the same
group or complex. Individuals exposed to M. bovis
(BCG, ATCC strain) may skin test convert although
no actual infectivity or disease occurs. Risk of
exposure would come from those mechanisms that
grind the waste. Mycobacterium terrae is equivalent
to M. tuberculosis in resistance to chemical
inactivation. In Europe it is recommended for
disinfectant testing. M. terrae does not grow as
rapidly as M. bovis or M. tuberculosis.

Both B. stearothermophilus and B. subtilis spores
are commonly used as biological indicators for both
thermal and chemical resistance. B.
stearothermophilus spores exhibit more thermal and
chemical resistance than spores from B. subtilis.

After discussion on the rationale for selection of the representative biological indicators presented
above, consensus by the committee was attained on recommending the use of these biological
indicator strains for treatment technology efficacy testing.

2.4 Quantification of Microbial Inactivation

Establishing the mechanisms to quantify the level of microbial inactivation is essential in
developing the format and requirements of the guidance protocols. As presented and discussed,
microbial inactivation ("kill") is equated to "Log, Kill" which is defined as the difference between
the logarithms of number of viable test microorganisms before and after treatment.
definition is translated into the following formula:

15


Dependent on the treatment process and efficacy protocols used, other factors may also influence
the evaluation results. As such, the committee could not define specific protocols, but

Ttecommended that protocols evaluating medical waste treatment systems specifically delineate
or incorporate:

. Waste compositions that typify actual waste to be processed; -
4
. Waste types that provide a challenge to the treatment process;
° Comparable conditions to actual use (i.e, process time, temperature,

chemical concentration, pH, humidity, load density, load volume);

. Assurances that biological indicators (ie. ampules, strips) are not
artificially affected by the treatment process;

. Assurances of inoculum traceability, purity, viability and concentration;

° Dilution and neutralization methods that do not affect microorganism
viability;

° Microorganism recovery methodologies that are statistically correct (i.e.,
sample collection, number of samples/test, number of colony forming
units/plate); and

° Appropriate microbial culturing methods (i.e., avoidance of microbial

competition, the selection of proper growth media and incubation times).

Based on the results obtained from challenge load testing, the medical waste treatment technology
may be limited in its application to not treating all categories or types of medical wastes.
Physical or aesthetic characteristics may also predicate the limitations applied or the conditions
of the equipment’s use. If certain medical waste categories are excluded from the treatment
process, the state should specify for the manufacturer (vendor) and the user of the equipment the
waste segregation parameters that will be employed to prohibit the waste from treatment and the
mechanisms of treatment/disposal to be utilized for these excluded wastes.

Consideration should also be given to the equipment’s use in a particular setting when applying
challenge load testing. The composition of the challenge load would be conceivably different and
more challenging if a particular application treats a medical waste stream containing a higher
proportion of a waste type or composition that is difficult to treat by that process. Conversely,
challenge loads for technologies whose primary application is hospital medical waste, might be
relaxed if that technology was applied only to waste generated by physician offices. Efficacy
testing protocols may also require modification dependent on the size or throughput of the
equipment. Multiple testing points might be required due to the waste volume processed or the
treatment process,

20


microorganisms required to demonstrate the prescribed Log reduction as defined in Level III (i.e.,
a 6 Log, reduction for vegetative microorganisms and a 4 Log,, reduction for spores). See
Appendix A (Section C3) and Appendix C for a detailed process description.

This step can be defined by the following equation:
LogRC = LogylC - LogipNR P

where:

LogiRC > 6 for vegetative microorganisms and > 4 for
bacterial spores;

Log,RC is the number of viable "Control" microorganisms
(in colony forming units per gram of waste solids) recovered
in the non-treated processed waste residue;

LogyoIC is the number of viable "Control" microorganisms
(in colony forming units per gram of waste solids)
introduced into the treatment unit; and

Log,oNR is the number of "Control" microorganisms (in
colony forming units per gram of waste solids) not
recovered in the non-treated processed waste residue.

Rearranging the equation above enables the calculation of microbial loss due to dilution, physicai
manipulation, or residue adhesion during the treatment process. Log,,NR represents an
accountability factor for microbial loss and is defined by the following equation:

LogygNR = Log,gIC - Log,RC.

The second step ("Test") is to operate the treatment unit as in the "Control" run with the selected
biological indicators, but with the addition of the microbial inactivation agent. After processing,
waste samples are collected and washed as in the "Control" to recover any viable biological
indicator organisms in the sample. From data collected from the "Test" and "Control", the level
of microbial inactivation (i.e, "Log, Kill") can be calculated by employing the following
equation:

Log, Kill = Log, IT - Log,)NR - LogiRT
where:
Log,cKill is equivalent to the term Logi, reduction;
Log,oIT is the number of viable "Test" microorganisms (in
colony forming units per gram of waste solids) introduced

into the treatment unit. Log,IT = Log, IC;

17


Log,oKill = Log,o(cfu/g Introduced) - Log,o(cfu/g Recovered)

where:

Log, Kill is equivalent to the term Log,, reduction;

"Introduced" is the number of viable test microorganisms
introduced into the treatment unit;

"Recovered" is the number of viable test microorganisms
recovered after treatment; and

“cfu/g" are colony forming units per gram of waste solids.

A Log, Kill of 6 or greater is equivalent or less than a one millionth [0.000001] survival
probability in a microbial population or a 99.9999% reduction or greater of that population.

Using the Level III definition recommended by the committee as shown in Table J, a Log, Kill
of 6 (e.g., 6 Log, reduction) is required of vegetative bacteria, fungi, lipophilic/hydrophilic
viruses, parasites, and mycobacteria and a Log, Kill of 4 (e.g., 4 Log,, reduction) is required of
B, stearothermophilus or B. subtilis spores. Employing the above equation to quantify microbial
inactivation will require the consideration of the methods of biological indicator introduction and
recovery. For those treatment processes that can maintain the integrity of the carrier (i.e.,
ampules, plastic strips) of the desired microbiological test strain, commercially available
biological indicators of the required strain and concentration can be easily placed, recovered, and
cultured to demonstrate efficacy. Quantification is evaluated by growth or no growth of the
cultured biological indicator. For example, if an ampule that contained 1 X 10° B.

stearothermophilus spores were treated, retrieved, and cultured, no growth would demonstrate a
4 Log, reduction.

For those treatment mechanisms that cannot ensure or provide integrity of the biological indicator
carrier, quantitative measurement of efficacy requires a two-step approach. The purpose of the
first step is to account for the reduction of microorganisms due to equipment design (i.e., dilution
of indicator organisms or physical entrapment).

This first step, the "Control", is typically performed using microbial cultures (i.e., liquid
suspensions) of predetermined concentrations necessary to ensure a sufficient microbial recovery
at the end of this step. The microbial suspension is added to a standardized surrogate medical
waste load that is processed under normal operating conditions without the addition of the
microbial inactivation agent (i.e., heat, chemicals). Standard loads may vary depending on the
various treatment challenges (i.e., high moisture content, high organic load, high density) required
of the equipment. After processing, waste samples are collected and washed to recover the
biological indicator organisms in the sample. Recovered microorganism suspensions are plated
to quantify microbial recovery. The number of viable microorganisms recovered serves as a
baseline quantity for comparison to ‘the number of recovered microorganisms from wastes
processed with the microbial inactivation agent. The required number of recovered viable
indicator microorganisms from the "Control" must be equal to or greater than the number of

16



3.0 PROCESS FOR APPROVING MEDICAL WASTE TREATMENT TECHNOLOGIES

State approval of an emerging medical waste treatment technology is necessary to ensure that the
technology can effectively and safely treat medical waste. From discussions, the completed
approval process can be viewed as fulfilling, where applicable, three components:

. Approval of the technology by the state to ensure the technology is
effective in safely inactivating microorganisms to specified criteria;

. Granting site approval to verify the sited equipment meets approved

specifications and efficacy requirements under actual operating conditions;
and

° USEPA FIFRA pesticide registration requirements, as applicable, for those

medical waste treatment technologies that use chemicals as the microbial
inactivator.

Each of these components requires information be supplied to states demonstrating that the
treatment technology is effectively treating medical waste by established criteria and that the
process is environmentally sound and occupationally safe. Information necessary for proper
review of medical waste treatment technologies is provided for each component described below.

3.1 Biological Inactivation Efficacy: Establishing Protocols

Methodology employed to determine efficacy of the technology will, by necessity, need to be
developed by the equipment manufacturer to assure the protocols are congruent with the
treatment method. Protocols developed for efficacy testing should incorporate recognized
standard procedures such as those found in Test Methods for Evaluating Solid Waste, Physical/

Chemical Methods and Standard Methods for the Examination of Water and Waste Water (see
Selected Bibliography).

In establishing testing criteria to evaluate efficacy, the composition of the waste load(s) tested
is critically important. Depending on the treatment mechanism, efficacy may vary with waste
load composition (i.e., organic content, density, moisture or liquid content). Although the
committee recognized that waste composition may affect efficacy results considerably,
establishing specific requirements for challenge loads for all existing, pending, and future
treatment technologies is not practical or necessarily all inclusive. The committee recommended
that the equipment manufacturer prescribe those types of medical wastes that present the greatest
challenge to efficacy of the equipment and present protocols that adequately evaluate efficacy
under normal operating conditions. On submittal for evaluation by the state, the manufacturer’s
prescribed waste types and testing protocols could be accepted or modified at the discretion of
the reviewing agency.

19


Level Il criteria provides a sufficient degree of microbial inactivation;

Level Ul criteria may conflict with lesser inactivation criteria already
defined by the state; and

Level w or IV criteria can be applied, if necessary, to those medical. owaste
streams requiring an additional margin of safety.

Pa

Arguments for recommending Level III criteria were as follows:

It was the consensus (not unanimous) of the committee that Level III be required of all emerging
medical waste technologies. The committee took the position that Level II criteria were to be
established as a benchmark and as such, were applicable to all medical waste treatment devices.
The committee realized that there might be circumstances under which a state may allow

Level Il criteria serve as a margin of safety from the variables inherent
in the treatment of medical waste (i.c., waste packaging, waste
composition, waste density, and factors influencing the homogeneity of the
treatment process);

Segregation of some medical waste categories (i.e. laboratory cultures)
requiring Level II treatment would be impractical if Level I criteria were
in effect;

Medical waste treatment equipment industry already achieves Level DI
criteria; and

Level II or Level IV criteria may still be allowed dependent on the
technology application or waste type processed.

Telaxation of the Level II requirement.

The committee rejected the allowance for exception to Level II standards for those technologies
that could be termed "counter-top" devices designed for a specific medical waste category.
Relaxation from Level II] to Level II criteria was not considered warranted on the basis of ‘2
equipment’s:

Inability to inactivate spores;
Designation as a small quantity treatment device;

Designation for treating minimally contaminated medical waste categories;
or

Exhibiting . difficulty to demonstrate microbial inactivation through
designated protocols (i.e., a needle thermal-destruction device).

11


LogjoNR is the number of "Control" microorganisms (in
colony forming units per gram of waste solids) not
recovered in the non-treated processed waste residue; and

Log,oRT is the number of viable "Test" microorganisms (in
colony forming units per gram of waste solids) recovered in .
treated processed waste residue. o

7

Appendix C (Section III) serves to illustrate the application of the equations presented above.

Formulas used in the discussion above for the quantification of microbial inactivation were
modified from those used by Illinois EPA in their final regulations (June 1993) entitled
“Potentially Infectious Medical Wastes" (see Selected Bibliography).

After discussion on the use and application of the formulas and calculations presented above,

consensus by the committee was unanimous on recommending the use of the formulas and
methods of calculation in the enumeration of medical waste treatment technology efficacy.

18


Bacterial Spores - B. stearothermophilus (ATCC 7953)
B. subtilis (ATCC 19659)

The committee recommended that one or more of the representative microorganisms from each
microbial group be used in efficacy evaluation. Specific criteria for the selection of these
microorganisms are provided below in Table II:

TABLE Ill - BIOLOGICAL INDICATOR SELECTION CRITERIA

Vegetative Bacteria - Staphylococcus aureus and Pseudomonas aeruginosa
were selected to represent both gram-positive and
gram-negative bacteria, respectively. Both are
currently required by the Association of Official
Analytical Chemists (AOAC) use-dilution method
and both have been shown to be resistant to
chemical inactivation.

Fungi - The selection of Candida albicans and Penicillium
chrysogenum was based on reported data indicating
these organisms representing yeast and molds,
respectively, are the most resistant to germicides.
Although Trichophyton mentagrophytes is the
AOAC test organism for molds, Penicillium
chrysogenum is reported to be more resistant to
germicides. The inclusion of Aspergillus niger as
an indicator organism was based on its familiarity as
acommon mold.

Viruses - Lipophilic (enveloped) viruses are less resistant to
both thermal and chemical inactivation than the
hydrophilic (nonenveloped) viruses. As such,
enveloped viruses such as HIV, Herpes simplex
virus and Hepatitis B virus are less resistant than
enveloped viruses such as Poliovirus, Adenovirus,
and Coxsackievirus. Polio 2 (attenuated vaccine
strain) and Polio 3 virus were selected based on
their relative higher chemical and thermal resistance.
Additionally, the use of an enterovirus (e.g., Polio 2
or Polio 3) can provide a stringent measure of
efficacy for irradiation treatment processes. MS-2
bacteriophage was selected as a Hepatitis virus
surrogate in that this bacteriophage offers a
comparable degree of chemical and thermal
resistance, is safe to handle and easy to culture.

14


. Meeting, where possible, the criteria established above;

. Representing, where possible, those organisms associated with medical
waste; and
. Providing a biological challenge equivalent to or greater than that

associated with microorganisms found in medical waste.

- Biological indicators selected to provide documentation of relative resistance to an inactivating
agent should be chosen after evaluation of the treatment process as it relates to the conditions
used during comparative resistance research studies described in the literature. Literature studies
support the assertion that the degree of relative resistance of a micrcorganism to an inactivating
agent can be dependent on various factors (i.e., pH, temperature). Conditions used in literature
studies that demonstrate a relatively high degree of resistance of a particular microorganism may
be significantly different to the conditions found within the treatment process. A comparison of
the conditions used in the literature to those used in the treatment process should be made to
determine if relative microbial resistance can be altered (i.e., lowered) as a result of treatment
process conditions.

The committee emphasized that although the microorganisms selected represent pathogen
surrogates, these selected surrogates may have the potential to be pathogenic under certain
conditions. As such, the committee recommended that all testing be conducted using recognized
microbial techniques. For those pathogen surrogates that still retain some higher degree of

pathogenicity (e.g., Cryptosporidium, Giardia, and Mycobacteria), efficacy testing should be
conducted only by qualified laboratory personnel.

TABLE Il - RECOMMENDED BIOLOGICAL INDICATORS

Vegetative Bacteria - Staphylococcus aureus (ATCC 6538)
Pseudomonas aeruginosa (ATCC 15442)

Fungi - Candida albicans (ATCC 18804)
Penicillium chrys chrysogenum (ATCC 24791)
Aspergillus niger

Viruses - Polio 2, Polio 3
MS-2 Bacteriophage (ATCC 15597-B1)

Parasites : Cryptosporidium spp. oocysts
Giardia spp. cysts

Mycobacteria : Mycobacterium terrae

Mycobacterium phlei
Mycobacterium bovis (BCG) (ATCC 35743)

13


The committee realized that there might be circumstances under which a state may allow
relaxation of the Level II requirement. These exceptions would by necessity need to be made
on a case-by-case basis, would require the equipment manufacturer to provide a rationale for
relaxation, and would require adequate supporting documentation to substantiate that rationale.

The committee also debated if laboratory wastes (i.e. discarded cultures and stocks of pathogenic
agents) should require sterilization (i.e. meet Level IV criteria) on the basis that these wastes may
contain high concentrations of known pathogens. The committee took the position that Level DI
criteria remained the standard for all medical waste categories. The committee emphasized,
however, that laboratories should be aware that cultures and stocks of disease-causing agents may
require sterilization before disposal. In addition to guidelines set by the Centers for Disease
Control in Biosafety in Microbiological and Eiomedical Laboratories, (1993) and standards of
the College of American Pathologists (CAP), some states require laboratory cultures to be
incinerated or autoclaved (i.e., steam sterilized) before leaving the laboratory or before being
disposed of. Although no specific recommendations for medical waste disposal are made under
the Ciimical Laboratory Improvement Amendments (CLIA), medical waste disposal practices are
receiving increased scrutiny during routine inspections.

2.3 Representative Biological Indicators

In the absence of an ultimate pathogen surrogate to represent all defined microbial groups, the
selection of pathogen surrogates representing vegetative bacteria, fungi, parasites, viruses,
mycobacteria, and bacterial spores was considered necessary to define and facilitate any state

approval process. Criteria defining surrogate selection should include that any surrogate
recommended:

. Not affect healthy individuals;

. Be easily obtainable;

. Be an ATCC registered strain, as available;
° Be easily cultured and maintained; and

. Meet quality control requirements.

Microorganism strains obtained from the American Type Culture Collection (ATCC) and methods
prescribed by the Association of Official Analytical Chemists (AOAC) assist in fulfilling these
recommendations by (1) providing traceable and pure cultures of known characteristics and
concentration and (2) providing recognized culturing protocols and detailed sampling and testing
protocols.

Provided in Table II are the biological indicators recommended by the committee for testing
microbial inactivation efticacy in medical waste treatment processes. The selection of these
representatives was based on each microorganism:

12