THE JOURNAL OF INFECTIOUS DISEASES • VOL. 134, NO.2· © 1976 by the University of Chicago. All rights reserved.
AUGUST 1976
Hepatitis B Surface Antigen in Environmental Samples from Hemodialysis Units From the Laboratory of Medical Microbiology and Hospital Epidemiology, and the Divisions of Hematology and Nephrology, Department of Medicine, University of Groningen, University Hospital, Groningen, The Netherlands
J. Dankert, J. Uitentuis, B. Houwen, A. M. Tegzess, and G. K. van der Hem
been taken for prevention of transmission of hepatitis B infection in dialysis units [3, 8, 11, 12]. To assess environmental contamination with HB s Ag in dialysis units, we investigated the presence of blood and HB s Ag on surfaces in two completely separated hemodialysis units. The investigated surfaces in both units were divided into five epidemiological categories, ranging from the patient and his immediate environment to surfaces with which the patient does not have direct physical contact. Samples from the surfaces were taken by means of previously moistened cotton swabs. After the swabs were rinsed, the rinse fluids were tested by radioimmunoassay (Ausria II, Abbott Laboratories, North Chicago, Ill.) for the presence of HB s Ag.
Outbreaks of infection with hepatitis B virus among patients and staff in renal hemodialysis units have been reported frequently [1-4]. Studies of the subtypes of hepatitis B surface antigen (HB s Ag) indicate that in dialysis units the infection might originate with one patient [5, 6]. The mode of spread of infection to other patients and staff is not fully understood. Pattison et al. [7] reported that infection is most commonly acquired by parenteral inoculation. Recently, Szmuness et al. [8] concluded that nonparenteral routes of infection probably also contribute to the spread of hepatitis B in dialysis units, and reservoirs of HB s Ag have been found to exist on various surfaces in dialysis units [9]. Since oral acquisition of the infection has been described [10], the presence of HB s Ag in environmental reservoirs may be responsible for the failure of the numerous measures that have
Materials and Methods
Patients and dialysis units. Two groups of nine patients from two separate hemodialysis units were included in the investigation. In unit A only HB s Ag-negative patients are treated, whereas in unit B 85 % of the patients are carriers of HB s Ag.
Received for publication August 20, 1975, and in revised form March 2, 1976. Please address requests for reprints to Dr. J. Dankert, Laboratory of Medical Microbiology and Hospital Epidemiology, Oostersingel 59, Groningen, The Netherlands.
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For assessment of environmental contamination with hepatitis B surface antigen (HBs Ag), the presence of blood and of HB s Ag was determined in samples from two completely separated hemodialysis units. Samples from mucosal and skin surfaces and samples of saliva from patients were also investigated. The surfaces of both units were divided into epidemiological categories to determine the distribution of environmental contamination with HB s Ag. HB s Ag was determined by radioimmunoassay. Oral samples from all patients seropositive for HB s Ag were positive for HB s Ag. The rectum and the skin were positive in three instances. The presence of blood did not coincide with HB s Ag. In the unit in which HB s Ag-positive patients were treated, 4.3 % of the total samples were HB s Ag-positive. Of the samples that were free of blood, 1.5% were HB s Ag-positive, whereas 18.5% of the bloodcontaminated samples contained the antigen. HB s Ag-positive samples were taken from surfaces in the immediate environment of HB s Ag-positive patients, as well as from surfaces with which patients had no direct physical contact. The data lead to the conclusion that spilled blood and hemodialysis equipment are not the only sources of contamination. This fact implies that HB s Ag-positive patients undergoing hemodialysis should be completely separated from HB s Ag-negative patients.
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Sample technique. Before sampling, a sterile cotton swab was moistened in a solution of sterile 1% bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, Mo.) in 0.9% NaCI (BSA-NaCI). Samples from the environmental surfaces were obtained by rubbing of a surface of 50-100 emwith these swabs. The swab was then placed immediately into a tube containing 1.5 ml of BSA-NaCI. Within 2 hr of taking the sample, the contents of this tube were mixed on a vortex mixer for 10 sec. The rinse fluid was poured into specimen vials, which were stored at - 20 C. The mucosa and the skin of the patients were sampled with cotton swabs as described. The rectal mucosa and the oral cavity (the gums, tongue, and pharyngeal area) were softly rubbed. A 50-cm2 surface of skin was sampled. Determination of RB s Ag. The collected fluid samples and specimens of serum and saliva were tested in duplicate for HB s Ag by solid-phase radioimmunoassay (Ausria-I1-125). A specimen was considered to be positive if in two consecutive assays the number of cpm was 2.1 times greater than the mean number of cpm obtained with negative controls of serum and with BSA-NaCI solutions. Each positive specimen was tested for specificity by a neutralization test with human serum containing anti-Hll, [13]. A sample was considered to be positive for HB s Ag if the prior addition of a fixed amount of urtlabeled human anti-Hls, resulted ina reduction of ~ 50% in the number of cpm. Detection of blood. During the environmental sampling the presence of visible blood was noted and recorded. For the demonstration of occult blood on the swabs, in the rinse fluid, and in saliva specimens, a guaiac test was applied. Results
Data on the presence of (occult) blood and of HB s Ag in samples taken from patients dialyzed in unit A (HB s Ag-negative) is presented in table 1, and data on patients treated in unit B (HB s Agpositive) in table 2. Blood was present in 13 (36.1 %) of 36 samples from nine patients in unit A as well as from eight patients in unit B. Blood was found mainly in saliva specimens and rectal mucosa samples. All samples collected from HB s
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Unit A, in use from 1966, has six hemodialysis stations. Approximately 36 hemodialyses are done each week. Unit B has 12 hemodialysis stations and has been in use since 1970; approximately 70 hemodialyses are carried out each week. In both units each patient is hemodialyzed for at least 20 hr per week; the disposable Gambro parallel flow dialyzer (Gambro AlB, Lund, Sweden) is used. Sterile disposable packs, lines, syringes, and needles are used in the dialysis procedure, and his or her own nondisposable items (bloodpressure cuff, stethoscope, etc.) are allocated to each patient. In almost all patients, access to the bloodstream is by means of a scarteriovenous fistula. A number of precautions are taken during the connection and disconnection of patients to and from the hemodialysis equipment. Staff members wear long-sleeved aprons, caps, masks, and gloves, which are all disposable. Since 1971 the patients and staff have been screened for HB s Ag and antibody to HB s Ag (anti-Hls.) at twoto four-week intervals. Aspartate aminotransferase (SGOT) and alanine aminotransferase (SG PT) were determined with the same frequency as was HB s Ag. Epidemiological categories in the dialysis units. Inclusion in category 0 indicated that the patient was the source of infection for his immediate as well as for his more remote environment. Specimens of serum and saliva, obtained after the patient had chewed sterile parafilm, were studied. Swab samples were taken from the oral cavity, the rectum, and the skin of the forearm that was not connected to the hemodialysis equipment. Category 1 included the environment in immediate association with the patient (bed, bed linen, floor surface under and beside the bed, stethoscope, blood-pressure cuff) . Category 2 consisted of items associated with the processing of blood samples taken from the patients (table surface, centrifuge, clotting-time testing apparatus, waste container). Category 3 comprised possible vectors, such as the trolley and the hands, gloves, or clothing of the staff. Category 4 consisted of the environment not in direct association with the patients, such as the desk and the telephone in the nurses' station, the handle of the refrigerator, and unused instruments.
HB s Ag in Hemodialysis Units
Ag-seronegative patients in both units were negative for HB s Ag. In contrast, HB s Ag was detected in 11 (39.3%) of 28 samples from the seven HB s Ag-positive patients in unit B. Samples most frequently found to be HB s Agpositive were those from the oral cavity, whereas specimens from the rectum and the skin were positive in only three instances. The presence of HB s Ag did not coincide with the presence of blood in the samples. No association was found between the presence of HB s Ag in samples from the mucosa of the oral cavity and the presence of this antigen in specimens of saliva. However, HB s Ag was detected in saliva and/or mucosal samples from all seven HB s Ag-positive patients. The results from the environmental survey in both dialysis units are recorded in table 3. As with the samples from patients, the proportion of environmental samples that contained blood was similar in both units. Blood was present in six (13.6%) of 44 environmental samples from unit
A and in 27 (16.6%) of 163 samples from unit
B. All samples taken from environmental surfaces in unit A were HB s Ag-negative, whereas seven (4.3 %) of 163 samples from unit B contained HB s Ag. Positive specimens in category 1 were taken from a table and from the floor near two beds in which HB s Ag carriers were dialyzed. In category 2 a positive sample was obtained from the table on which the clotting-time testing apparatus was located. The other HB s Ag-positive sample in this category was taken from the inside of the waste container. In category 3 a positive sample was collected from a nurse's glove, and in category 4 the door handle of a refrigerator was found to be HB s Ag-positive. The presence of blood on surfaces in unit B did not correlate exactly with the presence of HB s Ag. RB s Ag was found in five (18.5 %) of 27 blood-contaminated samples and in two (1.5%) of 136 samples with negative guaiac test results. Discussion
Favero et al. [14] have reported that contamination with HB s Ag can be assessed by use of a swab test in combination with a radioimmunoassay. In two hemodialysis units (A and B) we investigated, with use of these techniques, the presence of HB s Ag in oral, rectal, and skin samples from patients and in samples from environmental surfaces. In this survey patients and their hemodialysis apparatus in the units were considered as possible sources of infection [9, 15]. RB s Ag was present on mucosa and skin and in saliva of HB s Ag carriers treated in unit B. As reported by Heath-
Table 2. Presence or absence of hepatitis B surface antigen (HB s Ag) in relation to the presence of occult blood in specimens from seven HBs Ag carriers and two HBs Ag-negative patients who were dialyzed in unit B. Patient designation (category 0) Specimen J K L M N 0 P R Q Serum* H+B+ H-B+ H-B+ H+B+ H+B+ H+B+ H+B+ H+B+ H+B+ H-B+ H-B+ H+B+ H-B+ H+BH+BH-B+ Saliva H+B+ H+B+ H-BH-BH-BH-BH-BH+BOral mucosa H+B+ H+BH-B+ H-BH-BH-BH-BH+BH-B+ Rectal mucosa H+B+ H-B+ H-B+ H-BH-BH-BH-BH-BH-BH-BH-BH+BSkin NOTE. H+ = HB s Ag-positive; H- = HB s Ag-negative; B+ == occult blood-positive; B- = occult blood-negative. See text for definition of categories. * Guaiac test not performed.
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Table 1. Absence of hepatitis B surface antigen (HB s Ag) in relation to the presence of occult blood .in specimens of nine HBs Ag-negative patients hemodialyzed in unit A. Patient designation (category 0) Specimen A B C D E F G H I Serum Saliva + + + + + + + + Oral mucosa + Rectal mucosa + + + + Skin NOTE. A minus sign indicates that the patient's tests were negative for both HB s Ag and occult blood; a plus sign indicates that the patient was HB s Ag-negative but had a positive test for occultblood. See text for definition of categories.
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Table 3. The number of samples from environmental surfaces positive for hepatitis B surface antigen (HB s Ag) in relation to the presence of occult blood in hemodialysis units A (HB s Ag-negative patients) and B (HB s Ag-positive patients). No. of samples in epidemiological category*
Unit, presence of occult blood Unit A Occult blood-negative Occult blood-positive Total
90 (0) 12 (3) 102
3
8 (0)
5 (0) 0 5
2 (0) 10 5 (0) 12 (2) 17
10 (1) 0 10
4 5 (0)
0 5
31 (1) 3 (0) 34
* See Materials and Methods for definition of categories. t Numbers in parentheses denote the number of HB s Ag-positive samples.
cote et al. [16] and Villarejos et al. [17], oral specimens are frequently HBs Ag-positive. Samples from the rectal mucosa and the skin were much less frequently HB s Ag-positive. The low number of HB s Ag-positive samples from the rectal mucosa may be due to disintegration of the antigen [18] and to inhibitory effects of intestinal fluids on the determination of this antigen [19]. The presence of HB s Ag on the skin may result from secretion [20] as well as from environmental contamination. The results of the environmental survey show that HB s Ag could not be detected in dialysis unit A. In contrast, the antigen could be detected in 4.3% of the samples taken from environmental surfaces in unit B. Since there were no pronounced differences between units A and B in the incidence of blood in environmental samples, the presence of HB s Ag in samples from unit B indicates environmental contamination with this antigen. However, HB s Ag was recovered in only 18.5 % of the environmental samples contaminated with blood. This finding may be due to the limitations of a swab sample test in detection of microbial contamination, and it is obvious that the real incidence of surface contamination with HBs Ag may be higher than indicated by our results. For instance, it is known that the incidence of contaminants detected depends on the properties of the surface sampled [21]. In our survey HBs Ag could not be detected by the swab test in samples from textiles that were freshly contaminated with HB s Ag-positive blood. In contrast to this finding, the recovery of the antigen from stainless steel surfaces or from
glass slides contaminated with HBs Ag-positive blood was 100 % . The detection of contaminants from surfaces is also limited by the stability of the microbial agent. However, it is unlikely that this is an important factor in the recovery of the antigen since HBs Ag could be demonstrated in dried blood films on floor surfaces after two to three weeks at room temperature (authors' unpublished observation). In this survey five HBs Ag-positive samples from environmental surfaces were found in the vicinity of HB s Ag-positive patients and of the site where patients' blood samples are processed. One positive site was in an area to which the patients do not have direct access. The possibility of transmission by staff personnel is indicated by the detection of HB s Ag on a nurse's glove and the presence of the antigen on the door handle of a refrigerator. Airborne dissemination of HBs Ag has been suggested [17, 22] and should be taken into account as a possible mechanism of spread of infection. However, from the results of this survey, it seems that environmental contamination by HB s Ag-positive patients is rather limited and originates most frequently from the patient's direct vicinity. Moreover, it was noted that the samples from environmental category 1 of two HB s Ag-negative patients in unit B did not show the presence of HB s Ag. The results of this study indicate that environmental surfaces can be contaminated with HB s Ag and that environmental reservoirs can be established. Although the role of such reservoirs in the perpetuation of hepatitis B in dialysis units cannot be determined, this finding implies that
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Unit B Occult blood-negative Occult blood-positive Total
20 (O)t 4 (0) 24
2
HB s Ag in Hemodialysis Units
References 1. Jones, P.O., Goldsmith, H. J., Wright, F. K., Roberts, C., Watson, D. C. Viral hepatitis, a staff hazard in dialysis units. Lancet 1:835-840, 1967. 2. London, W. T., DiFiglia, M., Sutnick, A. 1., Blumberg, B. S. An epidemic of hepatitis in a chronichemodialysis unit. N. Eng!. J. Med. 281 :571-578, 1969. 3. Polakoff, S., Cossart, Y. E., Tillett, H. E. Hepatitis in dialysis units in the United Kingdom. Br. Med. J. 3 :94-99, 1972. 4. Bosch, E., Kolk-Vegter, A. J. Control of serum hepatitis in a dialysis unit. Neth. J. Med. 16:200-211, 1973. 5. Mosley, J. W., Edwards, V. M., Meihaus, J. E., Redeker, A. G. Subdeterminants d and y of hepatitis B antigen as epidemiologic markers. Am. J. Epidemiol. 95:529-535, 1972. 6. van Kooten Kok-Doorschodt, H. J., van den Akker, R., Gispen, R Determination and distribution of two types of hepatitis-associated antigen. J. Infect. Dis. 126: 117-122, 1972. 7. Pattison, C. P., Maynard, J. E., Berquist, K R, Webster, H. M. Serological and epidemiological studies of hepatitis B in haemodialysis units. Lancet 2: 172-174, 1973. 8. Szmuness, W., Prince, A. M., Grady, G. F., Mann, M. K, Levine, R W., Friedman, E. A., Jacobs, M. J., Josephson, A., Ribot, S., Shapiro, F. L., Stenzel, K. H., Suki, W. N., Vyas, G. Hepatitis B infection. A point-prevalence study in 15 US hemodialysis centers. J.A.M.A. 227:901-906, 1974.
9. Favero, M. S., Maynard, J. E., Petersen, N. J., Boyer, K. M., Bond, W. W., Berquist, K. R, Szmuness, W. Hepatitis-B antigen on environmental surfaces. Lancet 2: 1455, 1973. 10. Giles, J. P., McCollum, R W., Berndtson, L. W., Jr., Krugman, S. Viral hepatitis: relation of Australia/ SH antigen to Willowbrook MS-2 strain. N. Engl. J. Med. 281: 119-122, 1969. 11. Nordenfelt, E., Lindholm, T., Dahlquist, E. A hepatitis epidemic in a dialysis unit. Occurrence and persistence of australia-antigen among patients and staff. Acta Pathol. Microbiol. Scand. [B] 78: 692-700, 1970. 12. Hawe, B. J., Goldsmith, H. J., Jones, P. O. Dialysisassociated hepatitis: prevention and control. Br. Med. J. 1: 540-543, 1971. 13. Prince, A. M., Brotman, B., Jass, D., Ikram, H. Specificity of the direct solid-phase radioimmunoassay for detection of hepatitis-B antigen. Lancet 1: 1346-1350, 1973. 14. Favero, M. S., Bond, W. W., Petersen, N. J., Berquist, K R., Maynard, J. E. Detection methods for study of the stability of hepatitis B antigen on surfaces. J. Infect. Dis. 129:210-212, 1974. 15. Marmion, B. P., Tonkin, R W. Control of hepatitis in dialysis units. Br. Med. Bull. 28:169-179,1972. 16. Heathcote, J., Cameron, C. H., Dane, D. S. Hepatitis-B antigen in saliva and semen. Lancet 1:7173, 1974. 17. Villarejos, V. M., Visona, K. A., Gutierrez, A., Rodriguez, A. Role of saliva, urine and feces in the transmission of type B hepatitis. N. Engl. J. Med. 291: 1375-1378, 1974. 18. Moodie, J. W., Stannard, L. M., Kipps, A. The problem of the demonstration of hepatitis B antigen in faeces and bile. J. Clin. Pathol. 27:693-697, 1974. 19. Piazza, M., Di Stasio, G., Maio, G., Marzano, L. A. Hepatitis B antigen inhibitor in human faeces and intestinal mucosa. Br. Med. J. 2:334-337, 1973. 20. Teletar, H., Kayhan, B., Kes, S., Karacadag, S. HBAg in sweat. Lancet 2:461, 1974. 21. Hucker, G. J., Emery, A. J., Winkel, E. Adherence of film to plastics and china surfaces. Journal of Milk and Food Technology 14:95-99, 1951. 22. Almeida, J. D., Chisholm, G. D., Kulatilake, A. E., MacGregor, A. B., MacKay, D. H., O'Donoghue, E. P. N., Shackman, R, Waterson, A. P. Possible airborne spread of serum-hepatitis virus within a haemodialysis unit. Lancet 2: 849-850, 1971. 23. Gocke, D. J. Type B hepatitis-good news and bad. N. Eng!. J. Med. 291: 1409-1411, 1974.
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spread of HB s Ag in a dialysis unit cannot be prevented completely by use of disposable artificial kidneys and application of protective measures during direct contact with the patient or the hemodialysis equipment. Since oral acquisition of the infection has been proven [10] and minor skin abrasions may provide a portal of entry, exposure to environmental reservoirs may contribute to the occurrence of hepatitis B. Therefore, it seems likely that, as has been suggested by Hawe et al. [12] and by Gocke [23], segregation of HB s Ag-positive patients from HB s Ag-negative patients, by use of completely separated units with their own staff and equipment, would 'be of value in preventing the spread of HB s Ag.
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AUGUST 1976
Hepatitis B Surface Antigen in Environmental Samples from Hemodialysis Units From the Laboratory of Medical Microbiology and Hospital Epidemiology, and the Divisions of Hematology and Nephrology, Department of Medicine, University of Groningen, University Hospital, Groningen, The Netherlands
J. Dankert, J. Uitentuis, B. Houwen, A. M. Tegzess, and G. K. van der Hem
been taken for prevention of transmission of hepatitis B infection in dialysis units [3, 8, 11, 12]. To assess environmental contamination with HB s Ag in dialysis units, we investigated the presence of blood and HB s Ag on surfaces in two completely separated hemodialysis units. The investigated surfaces in both units were divided into five epidemiological categories, ranging from the patient and his immediate environment to surfaces with which the patient does not have direct physical contact. Samples from the surfaces were taken by means of previously moistened cotton swabs. After the swabs were rinsed, the rinse fluids were tested by radioimmunoassay (Ausria II, Abbott Laboratories, North Chicago, Ill.) for the presence of HB s Ag.
Outbreaks of infection with hepatitis B virus among patients and staff in renal hemodialysis units have been reported frequently [1-4]. Studies of the subtypes of hepatitis B surface antigen (HB s Ag) indicate that in dialysis units the infection might originate with one patient [5, 6]. The mode of spread of infection to other patients and staff is not fully understood. Pattison et al. [7] reported that infection is most commonly acquired by parenteral inoculation. Recently, Szmuness et al. [8] concluded that nonparenteral routes of infection probably also contribute to the spread of hepatitis B in dialysis units, and reservoirs of HB s Ag have been found to exist on various surfaces in dialysis units [9]. Since oral acquisition of the infection has been described [10], the presence of HB s Ag in environmental reservoirs may be responsible for the failure of the numerous measures that have
Materials and Methods
Patients and dialysis units. Two groups of nine patients from two separate hemodialysis units were included in the investigation. In unit A only HB s Ag-negative patients are treated, whereas in unit B 85 % of the patients are carriers of HB s Ag.
Received for publication August 20, 1975, and in revised form March 2, 1976. Please address requests for reprints to Dr. J. Dankert, Laboratory of Medical Microbiology and Hospital Epidemiology, Oostersingel 59, Groningen, The Netherlands.
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For assessment of environmental contamination with hepatitis B surface antigen (HBs Ag), the presence of blood and of HB s Ag was determined in samples from two completely separated hemodialysis units. Samples from mucosal and skin surfaces and samples of saliva from patients were also investigated. The surfaces of both units were divided into epidemiological categories to determine the distribution of environmental contamination with HB s Ag. HB s Ag was determined by radioimmunoassay. Oral samples from all patients seropositive for HB s Ag were positive for HB s Ag. The rectum and the skin were positive in three instances. The presence of blood did not coincide with HB s Ag. In the unit in which HB s Ag-positive patients were treated, 4.3 % of the total samples were HB s Ag-positive. Of the samples that were free of blood, 1.5% were HB s Ag-positive, whereas 18.5% of the bloodcontaminated samples contained the antigen. HB s Ag-positive samples were taken from surfaces in the immediate environment of HB s Ag-positive patients, as well as from surfaces with which patients had no direct physical contact. The data lead to the conclusion that spilled blood and hemodialysis equipment are not the only sources of contamination. This fact implies that HB s Ag-positive patients undergoing hemodialysis should be completely separated from HB s Ag-negative patients.
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Sample technique. Before sampling, a sterile cotton swab was moistened in a solution of sterile 1% bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, Mo.) in 0.9% NaCI (BSA-NaCI). Samples from the environmental surfaces were obtained by rubbing of a surface of 50-100 emwith these swabs. The swab was then placed immediately into a tube containing 1.5 ml of BSA-NaCI. Within 2 hr of taking the sample, the contents of this tube were mixed on a vortex mixer for 10 sec. The rinse fluid was poured into specimen vials, which were stored at - 20 C. The mucosa and the skin of the patients were sampled with cotton swabs as described. The rectal mucosa and the oral cavity (the gums, tongue, and pharyngeal area) were softly rubbed. A 50-cm2 surface of skin was sampled. Determination of RB s Ag. The collected fluid samples and specimens of serum and saliva were tested in duplicate for HB s Ag by solid-phase radioimmunoassay (Ausria-I1-125). A specimen was considered to be positive if in two consecutive assays the number of cpm was 2.1 times greater than the mean number of cpm obtained with negative controls of serum and with BSA-NaCI solutions. Each positive specimen was tested for specificity by a neutralization test with human serum containing anti-Hll, [13]. A sample was considered to be positive for HB s Ag if the prior addition of a fixed amount of urtlabeled human anti-Hls, resulted ina reduction of ~ 50% in the number of cpm. Detection of blood. During the environmental sampling the presence of visible blood was noted and recorded. For the demonstration of occult blood on the swabs, in the rinse fluid, and in saliva specimens, a guaiac test was applied. Results
Data on the presence of (occult) blood and of HB s Ag in samples taken from patients dialyzed in unit A (HB s Ag-negative) is presented in table 1, and data on patients treated in unit B (HB s Agpositive) in table 2. Blood was present in 13 (36.1 %) of 36 samples from nine patients in unit A as well as from eight patients in unit B. Blood was found mainly in saliva specimens and rectal mucosa samples. All samples collected from HB s
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Unit A, in use from 1966, has six hemodialysis stations. Approximately 36 hemodialyses are done each week. Unit B has 12 hemodialysis stations and has been in use since 1970; approximately 70 hemodialyses are carried out each week. In both units each patient is hemodialyzed for at least 20 hr per week; the disposable Gambro parallel flow dialyzer (Gambro AlB, Lund, Sweden) is used. Sterile disposable packs, lines, syringes, and needles are used in the dialysis procedure, and his or her own nondisposable items (bloodpressure cuff, stethoscope, etc.) are allocated to each patient. In almost all patients, access to the bloodstream is by means of a scarteriovenous fistula. A number of precautions are taken during the connection and disconnection of patients to and from the hemodialysis equipment. Staff members wear long-sleeved aprons, caps, masks, and gloves, which are all disposable. Since 1971 the patients and staff have been screened for HB s Ag and antibody to HB s Ag (anti-Hls.) at twoto four-week intervals. Aspartate aminotransferase (SGOT) and alanine aminotransferase (SG PT) were determined with the same frequency as was HB s Ag. Epidemiological categories in the dialysis units. Inclusion in category 0 indicated that the patient was the source of infection for his immediate as well as for his more remote environment. Specimens of serum and saliva, obtained after the patient had chewed sterile parafilm, were studied. Swab samples were taken from the oral cavity, the rectum, and the skin of the forearm that was not connected to the hemodialysis equipment. Category 1 included the environment in immediate association with the patient (bed, bed linen, floor surface under and beside the bed, stethoscope, blood-pressure cuff) . Category 2 consisted of items associated with the processing of blood samples taken from the patients (table surface, centrifuge, clotting-time testing apparatus, waste container). Category 3 comprised possible vectors, such as the trolley and the hands, gloves, or clothing of the staff. Category 4 consisted of the environment not in direct association with the patients, such as the desk and the telephone in the nurses' station, the handle of the refrigerator, and unused instruments.
HB s Ag in Hemodialysis Units
Ag-seronegative patients in both units were negative for HB s Ag. In contrast, HB s Ag was detected in 11 (39.3%) of 28 samples from the seven HB s Ag-positive patients in unit B. Samples most frequently found to be HB s Agpositive were those from the oral cavity, whereas specimens from the rectum and the skin were positive in only three instances. The presence of HB s Ag did not coincide with the presence of blood in the samples. No association was found between the presence of HB s Ag in samples from the mucosa of the oral cavity and the presence of this antigen in specimens of saliva. However, HB s Ag was detected in saliva and/or mucosal samples from all seven HB s Ag-positive patients. The results from the environmental survey in both dialysis units are recorded in table 3. As with the samples from patients, the proportion of environmental samples that contained blood was similar in both units. Blood was present in six (13.6%) of 44 environmental samples from unit
A and in 27 (16.6%) of 163 samples from unit
B. All samples taken from environmental surfaces in unit A were HB s Ag-negative, whereas seven (4.3 %) of 163 samples from unit B contained HB s Ag. Positive specimens in category 1 were taken from a table and from the floor near two beds in which HB s Ag carriers were dialyzed. In category 2 a positive sample was obtained from the table on which the clotting-time testing apparatus was located. The other HB s Ag-positive sample in this category was taken from the inside of the waste container. In category 3 a positive sample was collected from a nurse's glove, and in category 4 the door handle of a refrigerator was found to be HB s Ag-positive. The presence of blood on surfaces in unit B did not correlate exactly with the presence of HB s Ag. RB s Ag was found in five (18.5 %) of 27 blood-contaminated samples and in two (1.5%) of 136 samples with negative guaiac test results. Discussion
Favero et al. [14] have reported that contamination with HB s Ag can be assessed by use of a swab test in combination with a radioimmunoassay. In two hemodialysis units (A and B) we investigated, with use of these techniques, the presence of HB s Ag in oral, rectal, and skin samples from patients and in samples from environmental surfaces. In this survey patients and their hemodialysis apparatus in the units were considered as possible sources of infection [9, 15]. RB s Ag was present on mucosa and skin and in saliva of HB s Ag carriers treated in unit B. As reported by Heath-
Table 2. Presence or absence of hepatitis B surface antigen (HB s Ag) in relation to the presence of occult blood in specimens from seven HBs Ag carriers and two HBs Ag-negative patients who were dialyzed in unit B. Patient designation (category 0) Specimen J K L M N 0 P R Q Serum* H+B+ H-B+ H-B+ H+B+ H+B+ H+B+ H+B+ H+B+ H+B+ H-B+ H-B+ H+B+ H-B+ H+BH+BH-B+ Saliva H+B+ H+B+ H-BH-BH-BH-BH-BH+BOral mucosa H+B+ H+BH-B+ H-BH-BH-BH-BH+BH-B+ Rectal mucosa H+B+ H-B+ H-B+ H-BH-BH-BH-BH-BH-BH-BH-BH+BSkin NOTE. H+ = HB s Ag-positive; H- = HB s Ag-negative; B+ == occult blood-positive; B- = occult blood-negative. See text for definition of categories. * Guaiac test not performed.
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Table 1. Absence of hepatitis B surface antigen (HB s Ag) in relation to the presence of occult blood .in specimens of nine HBs Ag-negative patients hemodialyzed in unit A. Patient designation (category 0) Specimen A B C D E F G H I Serum Saliva + + + + + + + + Oral mucosa + Rectal mucosa + + + + Skin NOTE. A minus sign indicates that the patient's tests were negative for both HB s Ag and occult blood; a plus sign indicates that the patient was HB s Ag-negative but had a positive test for occultblood. See text for definition of categories.
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Table 3. The number of samples from environmental surfaces positive for hepatitis B surface antigen (HB s Ag) in relation to the presence of occult blood in hemodialysis units A (HB s Ag-negative patients) and B (HB s Ag-positive patients). No. of samples in epidemiological category*
Unit, presence of occult blood Unit A Occult blood-negative Occult blood-positive Total
90 (0) 12 (3) 102
3
8 (0)
5 (0) 0 5
2 (0) 10 5 (0) 12 (2) 17
10 (1) 0 10
4 5 (0)
0 5
31 (1) 3 (0) 34
* See Materials and Methods for definition of categories. t Numbers in parentheses denote the number of HB s Ag-positive samples.
cote et al. [16] and Villarejos et al. [17], oral specimens are frequently HBs Ag-positive. Samples from the rectal mucosa and the skin were much less frequently HB s Ag-positive. The low number of HB s Ag-positive samples from the rectal mucosa may be due to disintegration of the antigen [18] and to inhibitory effects of intestinal fluids on the determination of this antigen [19]. The presence of HB s Ag on the skin may result from secretion [20] as well as from environmental contamination. The results of the environmental survey show that HB s Ag could not be detected in dialysis unit A. In contrast, the antigen could be detected in 4.3% of the samples taken from environmental surfaces in unit B. Since there were no pronounced differences between units A and B in the incidence of blood in environmental samples, the presence of HB s Ag in samples from unit B indicates environmental contamination with this antigen. However, HB s Ag was recovered in only 18.5 % of the environmental samples contaminated with blood. This finding may be due to the limitations of a swab sample test in detection of microbial contamination, and it is obvious that the real incidence of surface contamination with HBs Ag may be higher than indicated by our results. For instance, it is known that the incidence of contaminants detected depends on the properties of the surface sampled [21]. In our survey HBs Ag could not be detected by the swab test in samples from textiles that were freshly contaminated with HB s Ag-positive blood. In contrast to this finding, the recovery of the antigen from stainless steel surfaces or from
glass slides contaminated with HBs Ag-positive blood was 100 % . The detection of contaminants from surfaces is also limited by the stability of the microbial agent. However, it is unlikely that this is an important factor in the recovery of the antigen since HBs Ag could be demonstrated in dried blood films on floor surfaces after two to three weeks at room temperature (authors' unpublished observation). In this survey five HBs Ag-positive samples from environmental surfaces were found in the vicinity of HB s Ag-positive patients and of the site where patients' blood samples are processed. One positive site was in an area to which the patients do not have direct access. The possibility of transmission by staff personnel is indicated by the detection of HB s Ag on a nurse's glove and the presence of the antigen on the door handle of a refrigerator. Airborne dissemination of HBs Ag has been suggested [17, 22] and should be taken into account as a possible mechanism of spread of infection. However, from the results of this survey, it seems that environmental contamination by HB s Ag-positive patients is rather limited and originates most frequently from the patient's direct vicinity. Moreover, it was noted that the samples from environmental category 1 of two HB s Ag-negative patients in unit B did not show the presence of HB s Ag. The results of this study indicate that environmental surfaces can be contaminated with HB s Ag and that environmental reservoirs can be established. Although the role of such reservoirs in the perpetuation of hepatitis B in dialysis units cannot be determined, this finding implies that
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Unit B Occult blood-negative Occult blood-positive Total
20 (O)t 4 (0) 24
2
HB s Ag in Hemodialysis Units
References 1. Jones, P.O., Goldsmith, H. J., Wright, F. K., Roberts, C., Watson, D. C. Viral hepatitis, a staff hazard in dialysis units. Lancet 1:835-840, 1967. 2. London, W. T., DiFiglia, M., Sutnick, A. 1., Blumberg, B. S. An epidemic of hepatitis in a chronichemodialysis unit. N. Eng!. J. Med. 281 :571-578, 1969. 3. Polakoff, S., Cossart, Y. E., Tillett, H. E. Hepatitis in dialysis units in the United Kingdom. Br. Med. J. 3 :94-99, 1972. 4. Bosch, E., Kolk-Vegter, A. J. Control of serum hepatitis in a dialysis unit. Neth. J. Med. 16:200-211, 1973. 5. Mosley, J. W., Edwards, V. M., Meihaus, J. E., Redeker, A. G. Subdeterminants d and y of hepatitis B antigen as epidemiologic markers. Am. J. Epidemiol. 95:529-535, 1972. 6. van Kooten Kok-Doorschodt, H. J., van den Akker, R., Gispen, R Determination and distribution of two types of hepatitis-associated antigen. J. Infect. Dis. 126: 117-122, 1972. 7. Pattison, C. P., Maynard, J. E., Berquist, K R, Webster, H. M. Serological and epidemiological studies of hepatitis B in haemodialysis units. Lancet 2: 172-174, 1973. 8. Szmuness, W., Prince, A. M., Grady, G. F., Mann, M. K, Levine, R W., Friedman, E. A., Jacobs, M. J., Josephson, A., Ribot, S., Shapiro, F. L., Stenzel, K. H., Suki, W. N., Vyas, G. Hepatitis B infection. A point-prevalence study in 15 US hemodialysis centers. J.A.M.A. 227:901-906, 1974.
9. Favero, M. S., Maynard, J. E., Petersen, N. J., Boyer, K. M., Bond, W. W., Berquist, K. R, Szmuness, W. Hepatitis-B antigen on environmental surfaces. Lancet 2: 1455, 1973. 10. Giles, J. P., McCollum, R W., Berndtson, L. W., Jr., Krugman, S. Viral hepatitis: relation of Australia/ SH antigen to Willowbrook MS-2 strain. N. Engl. J. Med. 281: 119-122, 1969. 11. Nordenfelt, E., Lindholm, T., Dahlquist, E. A hepatitis epidemic in a dialysis unit. Occurrence and persistence of australia-antigen among patients and staff. Acta Pathol. Microbiol. Scand. [B] 78: 692-700, 1970. 12. Hawe, B. J., Goldsmith, H. J., Jones, P. O. Dialysisassociated hepatitis: prevention and control. Br. Med. J. 1: 540-543, 1971. 13. Prince, A. M., Brotman, B., Jass, D., Ikram, H. Specificity of the direct solid-phase radioimmunoassay for detection of hepatitis-B antigen. Lancet 1: 1346-1350, 1973. 14. Favero, M. S., Bond, W. W., Petersen, N. J., Berquist, K R., Maynard, J. E. Detection methods for study of the stability of hepatitis B antigen on surfaces. J. Infect. Dis. 129:210-212, 1974. 15. Marmion, B. P., Tonkin, R W. Control of hepatitis in dialysis units. Br. Med. Bull. 28:169-179,1972. 16. Heathcote, J., Cameron, C. H., Dane, D. S. Hepatitis-B antigen in saliva and semen. Lancet 1:7173, 1974. 17. Villarejos, V. M., Visona, K. A., Gutierrez, A., Rodriguez, A. Role of saliva, urine and feces in the transmission of type B hepatitis. N. Engl. J. Med. 291: 1375-1378, 1974. 18. Moodie, J. W., Stannard, L. M., Kipps, A. The problem of the demonstration of hepatitis B antigen in faeces and bile. J. Clin. Pathol. 27:693-697, 1974. 19. Piazza, M., Di Stasio, G., Maio, G., Marzano, L. A. Hepatitis B antigen inhibitor in human faeces and intestinal mucosa. Br. Med. J. 2:334-337, 1973. 20. Teletar, H., Kayhan, B., Kes, S., Karacadag, S. HBAg in sweat. Lancet 2:461, 1974. 21. Hucker, G. J., Emery, A. J., Winkel, E. Adherence of film to plastics and china surfaces. Journal of Milk and Food Technology 14:95-99, 1951. 22. Almeida, J. D., Chisholm, G. D., Kulatilake, A. E., MacGregor, A. B., MacKay, D. H., O'Donoghue, E. P. N., Shackman, R, Waterson, A. P. Possible airborne spread of serum-hepatitis virus within a haemodialysis unit. Lancet 2: 849-850, 1971. 23. Gocke, D. J. Type B hepatitis-good news and bad. N. Eng!. J. Med. 291: 1409-1411, 1974.
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spread of HB s Ag in a dialysis unit cannot be prevented completely by use of disposable artificial kidneys and application of protective measures during direct contact with the patient or the hemodialysis equipment. Since oral acquisition of the infection has been proven [10] and minor skin abrasions may provide a portal of entry, exposure to environmental reservoirs may contribute to the occurrence of hepatitis B. Therefore, it seems likely that, as has been suggested by Hawe et al. [12] and by Gocke [23], segregation of HB s Ag-positive patients from HB s Ag-negative patients, by use of completely separated units with their own staff and equipment, would 'be of value in preventing the spread of HB s Ag.
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