Environ. Sci. Techno/. 1994, 28, 1767-1772
Study of Persistence of Enteric Viruses in Landfilled Disposable Diapers Mary S. Huber, Charles P. Gerba,. and Mortera Abbasradegant
Department of Soil and Water Science, University of Arizona, Tucson, Arizona 85721 Joseph A. Robinson The Upjohn Company, Kalamazoo, Michigan 49001
Susan M. Bradford
Orange County Water District, Fountain Valley, California 92728 Disposable diapers are one of many possible sources of infectious enteric viruses that are disposed of in landfills. A total of 218 disposable diapers were collected from 7 sites and 10 depths at three landfills. Of this total, 110 diapers were selected to be processed based on fecal content using a 1.5% beef extract elution, organic flocculationconcentration method to recover viruses. The concentrated samples were assayed on Buffalo Green Monkey (BGM) kidney cell cultures for the detection of enteroviruses and with cDNA probes specific for poliovirus, hepatitis A virus, and rotavirus. Enteroviruses were not detected in any sample assayed using cell culture techniques. Three samples were positive using nucleic acid probes for poliovirus while negative for rotavirus and hepatitis A. These results suggest that, although poliovirus RNA was present in some diapers, the viruses were not viable by cell culture assays after 2 years or longer in a landfill.
Introduction Approximately 200 million tons of municipal refuse are produced yearly in the United States, and approximately 67% of this solid waste goes into landfills (I). The physical composition of landfills is very heterogeneous and varies from city to city. Because of the heterogeneous composition of landfills, there is a diverse population of microorganisms, including viruses, associated with municipal solid waste. Viruses may be found in fecal material, of which there are many sources in landfills, some of which include pet feces, sewage sludge, septic tank waste, and disposable diapers (2). Disposable diapers make up 1-2 % by weight and 1-3 % by volume of landfills (3). These diapers may contain enteric viruses because infants and toddlers are often infected with enteric viruses and may excrete high numbers of enteric viruses in their feces. However, the relative contribution of disposable diapers to the total number of viruses in solid waste is estimated to be less than 1%of the viruses in the landfill from all sources ( 4 ) . When solid waste is placed in a landfill, it will begin to adsorb moisture depending upon the climatic conditions and the construction of the landfill. Leachate movement will not start until the solid waste has become saturated with water (field capacity). It is estimated that significant leachate movement will not occur for a t least 2-5 years after landfill closure for an average landfill (5). Once water
* Corresponding author.
+ Present address: American Water Works Service Co., Belleville,
IL 62221. 0013-936X/94/0928- 1767$04.50/0
0 1994 American Chemical Society
moves within the landfill, the resultant leachate can transport viruses outside the landfill in cases where the landfill is poorly constructed or unlined. It has been suggested that the low numbers of enteric viruses in landfill leachate may become a public health risk because eneteric viruses are transmitted by the fecal-oral route and thus can be transmitted by drinking contaminated groundwater (6). Enteric viruses can remain stable in the environment for months, and it is estimated that 1-10 infectious viral particles can initiate an infection (7). Few studies have been undertaken to determine the occurrence of viruses and their survival in disposable diapers in landfills and landfill leachate, but a recent review of the literature showed significant attenuation or inactivation of enteric viruses under landfill conditions and in leachates (5). The enteroviruses, especially polioviruses, are often present in the feces of infants and children in very high numbers even when there is no evidence of disease, due to the widespread use of the oral polio vaccine. The vaccine strains of poliovirus are commonly shed in the feces of vaccinated children for up to 2 months after vaccination (8). Rotavirus is the most common cause of gastroenteritis in children under 2 years of age (9) and is often present in the feces of infants and children. Hepatitis A virus is also a common infection in this age group, especially in day care centers. The hepatitis A virus is more resistant to inactivation a t high temperatures and is known to survive for prolonged periods in the environment (IO). However, a recent study found that hepatitis A virus persists no longer than poliovirus under simulated landfill conditions or in landfill leachates (11). Previous studies have found that 33% of disposable diapers collected from municipal solid waste were fecally soiled. Viable enteric viruses, poliovirus type 3, and echovirus 2 were recovered from 10% of the soiled diapers (12). Also, in a field study of leachate samples from 21 different landfills, poliovirus types 1and 3 were detected in 1 of 22 samples; however, this sample was from an improperly sealed landfill (13). These studies show that enteric viruses do enter landfills in disposable diapers and have some potential to be transported through the landfill and into the surrounding environment, but whether or not these organisms remain viable for prolonged periods of time in landfills is unknown. The purpose of this research was to study the occurrence of three enteric viruses in disposable diapers that had been buried in a landfill for a t least 2 years. Tissue culture assays using Buffalo Green Monkey (BGM) kidney cells were used to isolate enteroviruses because of the widespread occurrence of enterovirus infections and the wellEnvlron. Sci. Techno!., Vo!. 28, No. 9, 1994
1787
established methods for their isolation from environmental samples (14 ) . Each diaper sample was also examined using gene probes for the enteroviruses, rotavirus, and hepatitis A virus. Gene probes allow the detection of the presence of viral genomes whether the virus is viable or not.
Materials and Methods Sample Collection. Disposable babyimedicalladult diaper samples were collected from three landfills in the United States; one in the northeast, one in the southeast, and one in the southwest. Diapers were collected from several different sites and depths at each landfill. The sampling sites were chosen to include sections of the landfill that covered the entire age range of the landfill. A bucket auger was used to dig into each landfill site and bring a garbage sample to the surface where it was sifted through for disposable diapers (3). The temperature of each garbage sample was recorded, and the year the sample was placed in the landfill was determined from the dates on newsprint found in the garbage sample containing the diaper sample. Diapers were collected from landfill sites that had been active from 1980 to 1988. Each diaper sample was placed into a sealable plastic bag and labeled with the site number, depth, and age; placed on ice; and shipped to the laboratory where they were stored at 4 "C until processed (usually within 72 h). Sample Processing. All of the diapers that contained fecal material were processed as well as some that did not contain fecal material. Nonsoiled diapers were processed as a control to ensure that nothing in the diapers and/or processing was producing false positives. A variation of a method used to recover viruses from sludge (15)was used to recover viruses from diapers. Each diaper was placed into two 1-gal sealable plastic bags, one inside the other, with the diaper lining facing outward. One liter of sterile 1.5% beef extract (Beef Extract V, BBL; Benton Dickenson and Co., Cockeysville, MD), pH 9.5, was added to the bag. The diaper and beef extract were kneaded, by hand, for 5 min, and the pH of the eluent was adjusted to 7.2 using 1N HCl. The eluent was poured into autoclaved 250-mL centrifuge bottles and centrifuged a t 6000g for 10 min, the supernatant was removed with a pipet, and the pH was adjusted to 3.5. Organic flocculation, which involves viral adsorption to particulate solids, was used to concentrate viruses from the beef extract supernatant. The pH of the supernatant was lowered to 3.5, stirred for 10 min, and centrifuged at 16000g for 10 min at 4 "C. After centerifugation, the supernatant was discarded, and the protein pellet containing the viruses was resuspended in 0.15 M sodium phosphate buffer, pH 9.0 (15-20 mL) (16). The concentrate was mixed with an equal volume of Freon (1,1,2-trichlorotrifluoroethane, HPLC-grade; Aldrich, Milwaukee, WI), stirred for 30 min, and centrifuged at 16000g for 10 min, and the viralcontaining aqueous supernatant was decanted and saved. The lower Freon phase was discarded. Sephadex Columns. A 12.5-mL aliquot of each Freonextracted sample was layered ontoa 23-mL Sephadex G-50 (Pharmacia, LKB, Piscataway, NJ) column in a 60-mL Monoject syringe (Sherwood Medical, St. Louis, MO). If necessary, samples were diluted in 0.15 M sodium phosphate buffer, pH 7.0, so that 12.5-mL aliquots were always passed through the columns. The Sephadex G-50 was 1768
Environ. Sci. Technol., Vol. 28, No. 9, 1994
suspended in Tris buffer (pH 8.3) overnight and then autoclaved before use. The Sephadex column was used to reduce toxicity to the cell culture and polymerase chain reaction. The first 5-mL fraction from the column was discarded since it did not contain virus. The next 17.5mL fraction (the increase in volume is due to preexisting buffer in the column) was collected, and 0.5 mL each of kanamycin, gentamicin, mycostatin, and penicillin/streptomycin was added. All antibiotics were obtained from United States Biochemicals Corp., Cleveland, OH. The sample was incubated for 30 min in a 37 "C water bath. After each sample was treated with antibiotics, it was divided into two aliquots: one 12.5-mL aliquot to be assayed on BGM cells for viable enteroviruses and one 5-mL aliquot to be used for nucleic acid hybridization assays for the detection of enterovirus, rotavirus, and hepatitis A virus genomes. Each aliquot was frozen at -20 "C until assayed. Cell C u l t u r e Assays. Buffalo Green Monkey (BGM) kidney cells were used to assay the diaper samples for viable enteroviruses (14). The cells were grown in 75-cm2 plastic tissue culture flasks (Corning Glass Works, Corning, NY) with growth media containing 5 % fetal bovine serum. The growth media consisted of Eagle's minimum essential media (MEM) with Earles salts buffered with sodium bicarbonate (Mallinckrodt Inc., Paris, KY) and Hepes buffer (Research Organics, Cleveland, OH). The media also contains 200 mM glutamine (Fischer Scientific Co., Fair Lawn, NJ), penicillin/streptomycin (final concentration of 100 IU and 100pg, respectively, per 1mL of media), kanamycin 100 IU/mL, mycostatin of lo2 IU/mL, and approximately 5 5% of fetal bovine serum (Gibco Laboratories, Grand Island, NU). The cells were incubated a t 37 "C until a confluent monolayer was formed, usually within 5 days. After a confluent monolayer was formed, the growth media was poured off, and the cells were washed twice with prewarmed (37 "C) Tris- (Sigma Chemical Co., St. Louis, MO) buffered saline (63.2 g of Trizma base, 163.6 g of NaCl, 7.46 g of KCl, and 1.13 g of anhydrous NazH P 0 4 dissolved in 1600 mL of distilled water; final pH 7.2) (23). Five to seven aliquots (2-3 mL) of the concentrated sample were inoculated using 75-cm2tissue culture flasks. The flasks were incubated for 60 min at 37 "C and rotated every 15 min to allow virus adsorption to the cells. A total of 20 mL of MEM media was added to each flask. This was the same composition as the growth medium, except that it contained 2% fetal bovine serum and 50 pg of gentamicin/mL of media. The flasks were incubated at 37 "C for 14 days and checked for changes in cell morphology. The media in the flasks were removed on the seventh day of incubation, and fresh media were added. If greater than 90% of the monolayer was lost before the 14th day of incubation, the flasks were frozen at -20 "C and thawed a t 37 "C three times. A 1-mL sample from each thawed sample was inoculated onto new BGM monolayers as described above and monitored for 7 days for any changes in the cell morphology. The remaining 5 mL of the Freon-extracted Sephadextreated sample was stored a t -20 "C until it was assayed using a specific gene probe for each virus. Nucleic Acid Hybridization Assays. A poliovirus cDNA clone (PV104), consisting of base pairs 115-7440 inserted into the Pstl site of the plasmid pBR322, was provided by Drs. Rancaniello and Baltimore, Massachu-
setts Institute of Technology, Boston, MA (17). The hepatitis A virus cDNA clone (HAV-I), consisting of 1380 base pairs from the 3' end of the genome and inserted into the Pstl site of the plasmid pBR322, was provided by Dr. C. Wheeler, Center for Disease Control, Atlanta, GA (18, 19). The rotavirus probe was a clone of segment 4 (2500 bp) of human rotavirus Wa strain inserted into pUC13 from Dr. J . Flores, NIH, Bethesda, MD (20). All three plasmids were grown in transformed Escherichia coli HBlOl cells. Plasmid purification was done by cesium chloride centrifugation, phenol-chloroform extraction, and ethanol precipitation (21). Each vector and cDNA insert was labeled with both [32PldATP and [32P]dCTP [both with a specific activity of 3000 Ci/mmol (New England Nuclear, Boston, MA)] using nick translation (22). The average specific activity of each probe was 5.0 x 108 cpm/pg of cDNA. The radiolabeled cDNA was separated from free radionucleotides by chromatography through a Sephadex G-50 (Pharmacia, Piscataway, NJ) column and denatured by heating for 10 min in a boiling water bath (21). The Sephadex G-50 was suspended in Tris (pH 8.3) overnight and autoclaved before use. Five-fold dilutions of 1mL of each sample were made (0.2 mL of sample: 0.8 mL of HPLC grade water). Proteinase-K (Sigma, St. Louis, MO) was added at a final concentration of 100 pg/mL in the original sample and 50 pg/mL in each dilution tube. Proteinase-K degrades the viral capsid and releases viral nucleic acids. Each 1.5-mL polypropylene tube was incubated for 30 rnin a t 65 "C in a water bath (23). After incubation, the samples were placed on ice (no longer than 20 min), and centrifuged for 1min in a microfuge (DuPont, Wilmington, DE), and then the entire sample was spotted under vacuum onto a nylon membrane (Gene Screen Plus, DuPont, Boston, MA) using a dot-blot apparatus (MilliBlot-D, Millipore, Bedford, MA). The membrane was air-dried and baked in an oven (Napco, Portland, OR) for 2 h at 80 "C. The cDNA fragment within the vector (10 ng, 1ng, 0.1 ng) was used as a positive control on each membrane. HPLC-grade water (1mL, 100 pL, 10 pL) was used as the negative control. Each membrane also had a t least one sample that was negative by tissue culture as a second negative control. A 1-mL vol of all solutions used during the processing of the samples (distilled water, Tris buffer, 1.5% beef extract, 0.15 M Na2HP04, Freon) was tested for possible nonspecific binding with each cDNA probe. Hybridization and Washing Procedures. Prehybridization was done in a solution of 50% deionized formamide, 1%SDS, 5 % dextran sulfate, 5X SSPE buffer (0.75 M NaC1,0.05 M NaHZP04, 5 mM EDTA), and 45 pg/mL of sheared, denatured salmon sperm DNA (pH 7.4) (all from Sigma) for 2-4 h. The hybridization solution was the same as the prehybridization solution, except that the concentration of deionized formamide was decreased to 45% and the concentration of salmon sperm DNA was decreased to 2 pg/mL. After prehybridization, the membrane was placed into the hybridization solution, 10-20 ng of 32P-labeled probe was added to the sealable plastic bag, and hybridization was done for 24 h. After hybridization, the membrane was washed in 200 mL of buffer as follows: 2X SSC (standard saline-citrate, 0.15 M NaC1, 0.015 M sodium citrate, pH 7.0) a t room temperature for 5 rnin with constant agitation, 2X SSC and 1.0% SDS a t 50 "C for 30 min with constant agitation,
and 0.1x SSC at room temperature for 30 rnin with constant agitation (recommended washing procedure, Gene Screen Plus, Dupont, Boston, MA). The membrane was air-dried and placed on Kodak XAR-5 X-ray film (Eastman Kodak Co., Rochester, NY) with a DuPont Lightening-Plus (Wilmington, DE) intensifying screen for an exposure period of 48 h at -70 "C. RNase and DNase Reactions. When a positive signal was seen after hybridization, 1mL of the original sample was serially diluted (1:15) in distilled water, and 50 pL of proteinase-K (2 pg/pL) was added to each dilution tube. The sample was incubated for 30 rnin in a 65 "c water bath and then placed on ice for 20 min. Each sample was boiled for 5 min to denature the proteinase-K and cooled on ice, and 100 pg/mL of RNase A (Sigma) was added and incubated for 30 min in a 37 "C water bath. The samples were then spotted onto a membrane, hybridized, and put on X-ray film as previously described. Serial 5-fold dilutions of poliovirus (LSc-2ab) stock and poliovirus cDNA (1ng/pL) were also treated with proteinase-K and Rnase A as positive and negative controls. If a positive signal was seen after the RNase-treated sample was hybridized with a cDNA probe, the sample was treated with RQ1 DNAase (Promega, Madison, WI). DNase digests were done in 100-pL vol consisting of 50 mM Tris, pH 7.5, 10 mM MgC12, 20 pL of sample, 50 pg/ mL BSA, and 1 pL of DNase 1. The samples were incubated for 30 rnin in a 37 "C water bath. The reaction was stopped by adding 5 pL of 0.5 M EDTA. Sephadex Spin Columns. If a positive signal was seen after the RNase-treated sample was hybridized with a cDNA probe, 1 mL of the Freon-extracted sample was layered onto a Sephadex G-200 spin column (21) and centrifuged for 5 min a t 1600g to remove organic material. The sample was collected in a 1.5-mL tube, treated with proteinase-K as previously described, spotted onto a nylon membrane, hybridized with a cDNA probe, and put on X-ray film for 48 h. Preparation of Poliovirus Stocks. Poliovirus type 1 (LSc-2ab strain) was propagated on BGM cells. The cells were grown and maintained in Eagles MEM (minimum essential medium, Flow Laboratories, Irvine, CA) with Earles salts containing 5 % fetal bovine serum (HyClone Laboratories, Logan UT) by previously described methods (14). The viruses were inoculated onto cell monolayers grown in closed 32-oz glass bottles at a multiplicity of infection of 0.1. Virus stocks were harvested when >90% of the cell monolayer was destroyed. Harvesting consisted of freeze-thawing cells three times to liberate viruses from the cells, Freon extraction to remove lipids and disperse viral aggregates, and removal of cell debris by centrifugation in a Beckman (Palo Alto, CA) centrifuge at 15300g. The virus stocks were then dispensed in 1-mL aliquots and stored at 20 "C until used. Plaque Assay. Poliovirus stock was titered by the plaque overlay method (25),using BGM cells grown to confluency in plastic 6-well culture plates (Corning Glass Works) in a 5% COZincubator (VWR, Model 1810). Before sample inoculation, the growth media was removed from the cells, and 0.1 mL of serially diluted virus stock (0.1mL of virus:0.9 mL of Tris-buffered saline) was used to infect the cells. The viruses were allowed to adsorb to the cells for 45 min. After adsorption, an overlay of nutrient agar media was added. After 2 days, the agar overlay was removed, and the cell monolayers were stained Environ. Sci. Technol., Val. 28, No. 9, 1994
1760
Table 1. Number of Diapers Collected and Processed from Each Landfill
northeast southeast southwest total no. of diapers collected diapers processed diapers with fecal material processed
169
22 21 8
70 54
27
19 8
218 110 70
with 1.5% crystal violet for enumeration of plaques. Efficiency of Recovery of Viruses from Diaper Samples. Three experiments were conducted to determine the efficiency of recovery of poliovirus from the diapers. A new, unused diaper; a new diaper with 100 mL of leachate from the northeast landfill added and allowed to adsorb for 30 min at room temperature; and two diapers collected from the landfill, one with fecal material and one without, were assayed. To each of these diapers, 5 mL of poliovirus stock (LSc-2ab strain, 107 PFU/mL) was added and allowed to adsorb for 10 min a t room temperature. The seeded samples were then processed as described previously. After the elution, reconcentration, Freon extraction, and Sephadex G-50 treatments, a 0.6-mL sample was recovered, and 0.1 mL was assayed using the plaque overlay method as previously described. Seeded samples were processed using both 1.5% and 3% beef extract to determine which concentration of beef extract allowed better recovery of viruses. Both concentrations of beef extract gave similar results so the 1.5 % beef extract was used for processing.
Results A total of 218 disposable diapers were collected from the three landfills. Table 1 lists the number of diapers collected and processed from each landfill. The majority of diapers, 70 of the 110 diapers processed, were from the northeast landfill. Overall, 39 % of the diapers collected from the three landfills were fecally soiled. All of the soiled diapers were processed as well as some nonsoiled diapers. The majority of diapers were collected from garbage that had been placed in the landfills between 1984 and 1988 with the single largest number of diapers collected from 1984. The samples were collected from the landfill in 1990. All diapers were collected from depths of less than 30 ft. The temperatures of the garbage samples that diapers were collected from ranged from 21 to 68 "C. All of the processed samples that did not originally contain fecal material were negative by both cell culture assays and gene probe assays. The efficiency of the method developed to recover viruses from diapers was evaluated by inoculating new diapers and diapers collected from a landfill with high concentrations of poliovirus type 1. The number of plaqueforming units (PFU) of poliovirus added to each diaper was compared to the number of PFU recovered from each diaper after processing to determine the percent recovery. The efficiency of recovery averaged 28% for the diaper samples collected from the landfills (Table 2). The percent recovery of poliovirus 1 from diapers using two different concentrations of beef extract and the method developed in this study were evaluated. There was no difference in the recovery efficiency using the two different concentrations of virus. 1770 Environ. SCi. Technol., VOI. 28,
NO.
9, 1994
Table 2. Efficiency of Poliovirus Recovery from Diapers
type of diaper"
seeded diaper (PFU/mL)
final concn (PFUlmL)
%
recovered
new diaper 3.11 x 107 2.28 x 107 73 new diaper + 100 mL 2.50 X lo7 9.15 X 106 37 of leachate landfill diaper 1.12 x 107 3.18 x 106 28 Two different samples of each type of diaper was assayed, and the results were averaged.
To determine if any of the reagents or disposable diapers used in the recovery protocol were toxic to cell cultures or produced nonspecific binding with the gene probes, the protocol was followed without added virus and with new unused disposable diapers. The concentrated samples were inoculated onto BGM cells as well as assayed using each of the three gene probes. The cell monolayers remained intact during the 14-day monitoring period, and the samples did not produce a signal on X-ray film when assayed with the gene probes. In total, 110 diaper samples were assayed on BGM cells to detect enteroviruses. Of the samples processed, none were positive for enteroviruses using tissue culture methods. The diaper samples were also tested for viruses using cDNA gene probes specific for poliovirus, rotavirus, and hepatitis A virus. The sensitivity of each probe was determined by serially diluting the virus in distilled water, in 1.5% beef extract, and in a concentrated sample from the extraction procedure that was negative after an initial hybridization with each gene probe, spotting the samples onto a nylon membrane and hybridizing with each of the three probes. The poliovirus and hepatitis A probes were able to detect a minimum of 160PFU/mL in a concentrated sample, while the rotavirus probe was able to detect 4000 PFU/mL in a concentrated sample (Table 3). Fourteen samples gave a positive signal after being assayed with the poliovirus probe, and four samples were positive with the rotavirus probe. All of the positive samples were diapers from the northeast landfill. After confirmatory tests using RNase A, DNase, and Sephadex G-200 spin columns, six of the 14 signals were not seen after RNase A treatment and rehybridization. This indicates that the six samples may have contained viral RNA. If the original positive signal had been produced by RNA in the sample, treatment withDNase or Sephadex G-200 should not cause the signal to disappear. The combination of results indicating the possible presence of viral nucleic acid were only seen in three samples from the northeast landfill using the poliovirus cDNA probe (Table 4). The positive samples were no. 32, collected from a depth of 35 ft at 39 "C, placed in the landfill in 1984; no. 78, collected from a depth of 5 ft at 24 "C, placed in the landfill in 1982; and no. 47, which was a medical diaper, collected from a depth of 5 ft a t 31 "C,placed in the landfill in 1985. All three of the positive samples had been in the landfill for more than 4 years. Of the four samples initially positive by hybridization assay with the rotavirus cDNA probe, three still produced a positive signal after treatment with RNase A, and one gave a positive signal after treatment with DNase. These four signals disappeared after treatment with Sephadex G-200, indicating that it was not RNA in the sample that produced the initial signal but a compound that produced nonspecific binding with the rotavirus cDNA probe.
Table 3. Sensitivity*@of Virus cDNA Probes 5 x 106
1 x 105
2 x 104
+
+ + +
viral concn (PFU) 4 x 103 8 X lo2
+
+
1.6 X lo2
3.2 X loo
+
+ poliovirus in concentrate* + + f + + hepatitis A concentrate + + rotavirus in concentrated a This was determinedby serially diluting a known concentrationof each virus in water, beef extract, and a concentratedsample and assaying with the complementarycDNA probe. Concentrationsof cDNA of each probe (1 ng, 100 pg, 10 pg, or 1 pg) were positive controls. 1.5%beef extract and distilled water were negative controls. b The poliovirus cDNA probe is able to detect a minimum of 160 PFU/mL. The poliovirus 1 (Lsc) titer was 5 X 106 PFU/mL and was diluted in sample PG 102. c The hepatitis cDNA probe is able to detect a minimum of 160 PFU of hepatitis A virus/mL. The hepatitis A virus titer was 1 x 106 PFU/mL and was diluted in sample PG 108. The rotavirus cDNA probe is able to detect a minimum of 4000 PFU/mL. The rotavirus titer was 1.2 X lo5 PFU/mL and was diluted in sample PG 102. e Each cDNA probe was tested in triplicate.
+
Table 4. Detection of Enteroviruses in Diaper Samples from Northeast Landfill by Poliovirus cDNAa Hybridization and Tissue Culture Methods sample
Freon
32 47d
50 67 76
+ + + + + + + + + + + + + +
RNase
DNase
Sephadexb BGM assap
+ + + + + + + + +++ +
78 91 93 96 97 108 109d 110B 153 a 32P-labeledcDNA probe. G-200Sephadexcolumn. Cytopathic effect on BGM cells. d Adult or medical diaper.
Discussion Understanding the fate of enteric viruses in common landfilled items, such as disposable diapers, will be useful for a better understanding of how rapidly enteric viruses are inactivated in the landfill. This information will be important in improving our knowledge about the utility of a modern landfill to serve as an effective method of managing municipal solid waste. In order to interpret the significance of the observations obtained in this study, it is important to know the number of the disposable diapers which are landfilled that could contain enteric viruses. This number is difficult to determine because not every diaper contains fecal material and those that do contain fecal material do not all contain enteric viruses. For the purposes of this study, we have estimated the number of disposable diapers that contain enteric viruses. The number of viruses present in fecal material in diapers varies greatly and depends on factors such as the level of hygiene of the population, the prevalence of infection in the community, the season, the climate, and the population density. Any findings of enteric viruses in diapers recovered from a landfill must be interpreted relative to the expected rates of infection. The incidence of enterovirus infection in children 0-5 years of age in the United States has been reported to range from 2.4% to 14% (26). The incidence of rotavirus infection has been reported to range from 70% for hospitalized neonates to 26 5% for hospitalized children 7-24 months old (27, 28). The average annual rate of
rotavirus gastroenteritis is 10.4%for children under 2 years of age (29,30). The incidence of clinical hepatitis in the United States is 0.01% (31), but it is believed this represents only 15 % of the actual cases (32). This would make the actual incidence about 0.1% This may be an underestimation of the actual incidence of rotavirus and hepatitis A infections because both are often asymptomatic in children (33,34). For this study, it was assumed that the incidence of infection with enteric viruses in children in the United States is 10% (35). It is important to know if enteric viruses survive over time under the conditions in an acutal landfill in order to determine how long disposable diapers potentially serve as a source of infectious virus. Any disposal method for untreated domestic fecal material that presents a risk of microbial contamination of groundwater supplies should be carefully monitored. Cell culture assays using BGM cells were done to test for viable enteroviruses because it has been shown that BGM cells are the most sensitive for the detection of enteroviruses in environmental samples (36). Poliovirus would be the virus most commonly found in the feces of infants and toddlers due to the fact that most children under 2 years of age are vaccinated with the live polio vaccine three times within the first 2 years of life and shed the vaccine strains of poliovirus in their feces for several weeks after vaccination (8, 26). Viable enteroviruses were not detected in any of the 110 diaper samples using cell culture assays. The absence of viable enteroviruses is probably due to the time the diapers had been buried in the landfills and the temperatures that they had been exposed to in the landfills (37, 38). It is also possible that viable enteroviruses may not have been found in any of the diapers because the population may not have been excreting the viruses. Using binomial analysis (35,39) and the following formula
.
where p is the probability of recovering viruses from a diaper, n is the number of diapers processed that contain feces, and a is 0.05. Assuming only the soiled diapers could potentially harbor pathogens, the unknown true proportion of diapers potentially contaminated could be as high as 11 % (p = 0.05) (35). Of the 70 processed diapers that were fecally soiled, 11 % or eight diapers potentially contained pathogenic viruses and could be positive by tissue culture or gene probe methods. Theoretically, eight diapers should have been positive using gene probes, but only three were positive. Environ. Sci. Technol., Vol. 28, No. 9, 19S4
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The ability of our methods to detect viruses can be estimated using the recovery factor of 28% found in this study and considering the high number of viruses shed by infected individuals (103-1010 PFU) (22). This means that 102-109 PFU of viruses could be recovered from the samples, and these high numbers of viruses are easily detectable by the methods used. After confirmatory tests with RNAase A, DNase, and Sephadex G-200 spin columns, three of the 110 diaper samples were found to be positive using the poliovirus cDNA probe, while none of the samples were positive using the rotavirus and or hepatitis A virus probes. All three positive samples had been buried in a landfill for longer than four years, and all three were from the northeast landfill, which was probably due to the fact that the largest number of diaper samples were collected from this landfill. There was no correlation between depth, temperature, date of disposal, and a positive hybridization assay. The results of this study indicate that, even though viral RNA remains intact in diapers over time, the diapers are not unique sources of infectious viruses after they have been in landfills for longer than 2 years even though the viral nucleic acid may be detectable after several years. This study suggests that diapers do not require special handling (e.g.,to remove feces or inactivate viruses) before they are placed in landfills. It seems that the physical and chemical processes occurring in a landfill after at least 2 years are sufficient to inactivate viruses that may be present in the fecal material below the detection limits of the methods used in this study. Also, if a landfill is properly constructed, it is an acceptable means of disposal of disposable diapers, and thus the risk of groundwater contamination by enteric viruses in the fecal material is very low.
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Abstract published in Advance ACS Abstracts, August 1,1994.
