Vol. 56, No. 1

0099-2240/90/010264-05$02.00/0 Copyright © 1990, American Society for Microbiology

Model for Inactivation and Disposal of Infectious Human Immunodeficiency Virus and Radioactive Waste in a BL3 Facility MARGARET C. STINSON,'* MITCHELL S. GALANEK,1 ALAN M.



Environmental Medical Service, Massachusetts Institute of Technology, 20B-238,1 and The Whitehead Institute for Biomedical Research,2 Cambridge, Massachusetts 02139, and Infectious Disease Unit, Medical Service, Massachusetts General Hospital, Boston, Massachusetts 021143 Received 19 February 1989/Accepted 25 October 1989

A method is described for autoclaving low levels of solid infectious, radioactive waste. The method permits steam penetration to inactivate biologic waste, while any volatile radioactive compounds generated during the autoclave process are absorbed. Inactivation of radiolabeled infectious waste has been problematic because the usual sterilization techniques result in unacceptable radiation handling practices. If autoclaved under the usual conditions, there exists a high probability of volatilization or release of radioisotopes from the waste. This results in the radioactive contamination of the autoclave and the laboratory area where steam is released from the autoclave. Our results provide a practical method to inactivate and dispose of infectious radioactive waste. For our research, Bacillus pumilus spore strips and vaccinia virus were used as more heat-resistant surrogates of the human immunodeficiency virus (HIV). These surrogates were used because HIV is difficult to grow under most conditions and is less heat tolerant than the surrogates. In addition, B. pumilus has defined cell death values, whereas such values have not been established for HIV. Both B. pumilus and vaccinia virus are less hazardous to work with. The autoclave method is time efficient and can be performed by laboratory personnel with minimal handling of the waste. Furthermore, waste site handlers are able to visually inspect the solid waste containers and ascertain that inactivation procedures have been implemented.

As many as 365,000 cases of acquired immunodeficiency syndrome are expected in the United States by 1992 (6). Many more people will harbor the virus, and concentrated research efforts will continue to be directed towards preventing the spread of infection and the development of disease in infected individuals. The etiologic agent of the acquired immunodeficiency syndrome is the human immunodeficiency virus (HIV), previously designated as human T-lymphotropic virus type III or lymphadenopathy-associated virus (2, 4). Cumulative epidemiologic data indicate that HIV is transmitted almost exclusively by blood or blood products in vivo or via sexual excretions (7). HIV can also be transmitted in laboratory research settings, even in the absence of accidental needle sticks (13). Probing the mechanisms of HIV infection, replication analysis, vaccine research, and the development of effective HIV treatment often require the use of radioactive tracers. A management program for mixed infectious and radioactive waste must assure complete inactivation of HIV before disposal as radioactive waste. Infectious material is inactivated by one of several techniques. These include chemical and physical inactivation methods such as heat and steam penetration. The technique used to inactivate the infectious radioactive waste is determined by the physical form of the waste. The proposed technique is for solid waste only. Radioactive waste disposal personnel are not trained to handle infectious organisms, and they are often socially resistant to accepting this additional type of risk. For social, health, and regulatory reasons, it is crucial then that any infectious radioactive material first be biologically inactivated. In addition, waste handlers must be able to visually *

inspect the waste containers on site and ascertain that inactivation procedures have been implemented. Available laboratory techniques, such as monitoring reverse transcriptase activity, are not amenable to application by waste disposal personnel. The goal of our research was to demonstrate whether radioactive compounds, volatilized by routine autoclave procedures, can be completely contained within a primary enclosure (autoclave bag). Second, can the complete inactivation of HIV or more heat-resistant organisms such as Bacillus pumilus be assured by visual inspection of the autoclaved bag? MATERIALS AND METHODS Typical radionuclides used in HIV facilities are: inorganic 32P04- and 32P-labeled nucleoside triphosphates used in the end labeling of DNA, [35S]methionine and [35S]cysteine used in the metabolic labeling of proteins, 3H in the form of [3H]TTP used in reverse transcriptase assays, and 51Cr in the form of Na2CrO4 used in Cr release assays and cytotoxic T-lymphocyte assays. Work procedures involve the implementation of standard radiation protection precautions such as contamination control monitoring and dose reduction

techniques. Barriers required to contain volatile radioactive compounds were determined by an analysis for volatile radioactive compounds generated by an open bag autoclave system and measurement of containment provided by single- and double-barrier systems. Six radionuclides were studied: 3S, 32p, 125I, 3H, 14C, and 51Cr. Noninfectious wastes consisting of bench paper, gloves, pipette tips, and Eppendorf tubes in clear, transparent polypropylene autoclavable waste bags were studied. Open waste bags were autoclaved for 30 min at 121°C. These bags (Clavies; Bel-Art Products, Pequannoc, N.J.) are steam autoclavable to 121°C and are 0.04 mm thick.

Corresponding author. 264

VOL. 56, 1990

The open bag assured steam penetration throughout the waste. The autoclave used was manufactured by American Sterilizer Co., Erie, Pa. An air filter monitored all steam released from the autoclave throughout its operation. This was a high-volume air sampler produced by BGI Products, Waltham, Mass. Wipe tests were taken over several 100-cm2 areas after each run. The wipe locations were: the steam release area, the interior sides, top, and bottom of the autoclave, and the top surface of the waste bag. The air filter and wipes were analyzed for contamination by using liquid scintillation counting. In cases where the autoclave was found to have been contaminated, it was cleaned with absorbant towels and a "lift-away"-type foam made specifically for radioactive contamination removal. Cleaning was continued until wipes of the affected area showed no removable radioactive contamination. A single barrier system was then studied. Identical isotopes and procedures were followed as stated above with the exception that the waste bag was twist knotted and completely sealed with autoclave tape. The air filter and wipes were analyzed as described above. Two trial autoclave runs were conducted per isotope at 121°C. Mock runs were also conducted without the use of radioactive material to determine the exposure time and steam penetration conditions required to completely inactivate B. pumilus in a double-barrier system by using Diack autoclave controls (Diack, Inc., Beulah, Mich.) and temperaturesensitive autoclave tape. Heat and steam penetration for inactivating the virus were tested with two complementary tests. A double-barrier waste containment system with Diack controls taped on the inside of the inner waste bag was used. A Diack control was also taped to the outside of the outer waste bag. The Diack control pellet melts and changes color (from a dark blue to red) when a temperature of 121°C has been reached. These responses were first validated under known temperature conditions. A primary polypropylene bag containing the noninfectious, nonradioactive waste was contained within a second polypropylene bag. One pint (ca. 0.473 liters) of Speedi-Dri absorbant (Clint Sales, Beverly, Mass.) and various amounts of water were added to the inner waste bag to determine requirements for steam generation within the bag. Both the temperature-sensitive autoclave tape and Diack controls were taped inside the inner waste bag. This tape was placed in 3-in. (7.62-cm) strips throughout the interior of the inner bag. Autoclave tape was also used to seal the outer waste bag. Color changes and melting of the Diack pellet were monitored for the interior and exterior Diack controls. Color changes were also monitored for the autoclave tape. Both the Diack controls and autoclave tape were monitored under various water and exposure time conditions. Exposure times of 20, 30, and 40 min were used. The autoclave temperature remained constant throughout at 121°C. Three independent, identical trials were conducted for each condition tested. A method for suitable charcoal filtration was analyzed. The purpose of the filter is to contain radioactivity while permitting steam penetration and pressure equalization. The charcoal autoclave filter was constructed as follows. Five grams of activated charcoal was deposited in a drying tube (glass wool at each end of the drying tube contained the charcoal). The drying tube was a polyethylene tubular body with two serrated tips (the body had a 16-mm inner diameter and a 19-mm outer diameter; the tips accepted 6.4- to 9.5-mm inner diameter tubing). Two 12-in. (30.48-cm) by



24-in. (60.96-cm) autoclavable polypropylene bags were used to contain the radioactive waste. The final system was constructed as follows. A double bag was created by placing one polypropylene bag within a second bag. The bags were then filled with dry solid waste weighing an average of 500 g. Absorbant (1 pt) and water (2 ml) were added to the inner waste bag. The charcoal autoclave filter was tightly secured in place at the opening of the bags by autoclave tape, forcing all air flow in or out of the bag through the filter. Each radionuclide was tested in 10 independent trial runs. Air sample analyses and wipe tests were


In addition, 5 cm of rubber tubing was secured to the outer tip of the charcoal autoclave filter tube. Tubing was attached to an additional polyethylene filter tip containing glass wool fiber and a 1-cm2 charcoal filter. This additional filter permitted the direct sampling of air emerging from the charcoal filter drying tube. Individual waste runs were performed by using six radionuclides under conditions previously described. Following the autoclave runs, glass wool fibers and the charcoal filter were analyzed for contamination by using liquid scintillation counting. Finally, exposure time and steam penetration conditions were verified by seeding nonradioactive, noninfectious waste bags with B. pumilus spore strips (North American Science Associates, Inc., Northwood, Ohio). Spore strip performance data were assayed for a population of 1.6 x 105 spores per strip and a D value of 0. 15 min in steam at 121°C. Three spore strips were placed in each double bag waste system containing solid dry waste, 2 ml of water, and 1 pt of absorbant. Strips were distributed throughout the load with consideration given to difficult-to-sterilize locations. Nonautoclaved spore strips were used as positive controls. Both control and experimental B. pumilus spore strips were incubated for a period of 7 days at 37°C in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) in three separate trials. Spore strips were monitored for spore growth throughout the incubation period. To ensure that radiation emanating from the radioactive waste would have no effect on the inactivation requirements of B. pumilus, an additional six experimental runs were performed. The same procedures were then repeated with the addition of one radionuclide per run. The procedures used to inactivate B. pumilus were tested by using vaccinia virus. Vaccinia strain WR was used to infect confluent monolayers (25 cm2) of CV-1 cells grown in minimal essential medium (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 10% fetal bovine serum and glutamine. Portions (0.5 ml) of a vaccinia virus stock (approximately 108 PFU/ml) were aliquoted into three 3.7-ml glass screw-capped vials and placed inside the waste system, as previously described, and autoclaved. After the vials cooled, the autoclaved virus stock was added to the monolayers, and the cells were incubated at 37°C. As a control, 0.5 ml of live vaccinia virus from the same stock was added to a fourth flask (with a multiplicity of infection equal to 5 PFU per cell) and incubated with the autoclaved vials. RESULTS

Radioactive components were volatilized under the autoclave operating conditions (Table 1). Both 35S and 3H radioactive contaminants were observed under open bag conditions. Further autoclaving studies were done with completely sealed polypropylene bags containing noninfectious radioactive waste. Volatile radioactive 35S was able to




TABLE 3. Required exposure times and moisture conditions for doubly enclosed systems

TABLE 1. Open bag autoclave contamination Contamination"





0.5 15.2 86.0 0.3 1.3 5.0 3.0 60.0





3H 14C 5tCr


(dpm/100 cm2)

Color change in interior": Diack Tape

Air sample (dpm/m3)

Vol of water (ml)

Model for inactivation and disposal of infectious human immunodeficiency virus and radioactive waste in a BL3 facility.

A method is described for autoclaving low levels of solid infectious, radioactive waste. The method permits steam penetration to inactivate biologic w...
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