Hepatitis B surface antigen (HBsAg) infection in a hemodialysis unit. II. Factors affecting host immune response to HB8Ag Dharmendra P.S. Sengar, ph d; Abdur Rashid, md, frcp[c]; William A. McLeish, md, Jules E. Harris, md, frcp[c]; Roger A. Couture, md, frcp[c]; Monique Sutherland, lt

Summary: Serum from 86 hemodialysis patients, 105 healthy hospital staff "at risk" and 160 regular hospital staff was screened for hepatitis B surface antigen (HBsAg) and antibody (anti-HBs). The combined prevalence of HBsAg and anti-HBs was higher in the staff of the artificial kidney unit (57.7%) than in the hemodialysis patients (33.7%). The healthy subjects with HBsAg infection responded significantly more often by producing anti-HBs compared with the hemodialysis patients. Twelve of 29 (41.4%) hemodialysis patients with HBsAg infection produced anti-HBs, while 17 (58.6%) remained positive for HBsAg. This differential response could not be attributed to age,

spent undergoing hemodialysis, delayed cutaneous reactivity or response to phytohemagglutinin (PHA) or pokeweed mitogen (PWM). However, a much larger proportion of patients with HBsAg than with anti-HBs had previously received blood transfusions (88.2% v. 33.3%). Our results indicate that development of the chronic HBsAg carrier state or production of anti-HBs in uremic patients may be influenced by the route of immunization or the dose of antigen, or both. Although uremic patients

sex, time

maintain normal in vitro response to PHA and PWM, may have depressed immunity in vivo because of

they

decreased total number of

a

T-lymphocytes.

Resume: Linfection par antigene de surface de I'hepatite B (AgHBs) dans un service d'hemodialyse. II. Les facteurs qui affectent la reaction immunitaire de I'hote a l'AgHBs L'antigene de surface de I'hepatite B (AgHBJ et son anticorps (anti-HBs) ont ete recherches dans le serum de 86 hemodialyses, de 105 membres du personnel soignant, apparemment sains mais soumis a un "risque eleve" et de 160 membres du personnel hospitalier regulier. La prevalence combinee de l'AgHBs et de l'anti-HBs etait plus forte parmi le personnel du service de rein artificiel (57.7%) que chez les hemodialyses (33.7%). Les sujets sains mais infectes par l'AgHBs ont reagi significativement plus souvent par la production d'anti-HBs que les hemodialyses. Douze des 29 hemodialyses (41.4%) infectes par l'AgHBs ont produit des anticorps, tandis que 17

(58.6%) demeuraient positifs pour l'AgHBs. Cette

frcp[c];

difference de reaction etait independante de I'age, du sexe, de la duree du temps passe a I'hemodialyse, de la reactivite cutanee retardee ou de la reaction a la phytohemagglutinine (PHA) ou au mitogene extrait du raisin d'Amerique (PWM). II faut cependant noter qu'un nombre beaucoup plus grand de porteurs de I'antigene que de porteurs de I'anticorps avaient recu des transfusions sanguines auparavant (88.2% v. 33.3%). II ressort de nos resultats que I'apparition de I'etat chronique de porteur d'AgHBs ou la production d'anti-HBs chez les uremiques peut etre influencee par la voie d'immunisation ou par la dose d'antigene, ou par les deux facteurs. Meme si les uremiques maintiennent une reaction in vitro normale a la PHA et au PWM, ils peuvent presenter une immunite deprimee in vivo, en raison d'une diminution globale du nombre de lymphocytes T.

Hepatitis B surface antigen (HBsAg) infection has been re¬ ported to be frequent at various hemodialysis centres.1'13 It has been commonly observed that hemodialysis patients may show HBS antigenemia for several months, often with¬ out clinical symptoms of hepatitis. In contrast, healthy persons "at risk" show transient antigenemia, which often results in severe clinical hepatitis.2,10'11 Blumberg, Sutnick and London2 have hypothesized that the persistent anti¬ genemia in uremic patients may be due to a depressed immune state. A chronic HBsAg carrier state has also been postulated to be due to poor T-lymphocyte recognition of the infective agent.14 Recently a preponderance of HL-A3, W1915 and Sabell antigens16 has been reported in healthy HBsAg carriers. However, not all HBsAg carriers possess these antigens, which suggests that certain other genetic or environmental factors, or both, may also be related to the lack of immunity to HBsAg in the chronic carrier. We have studied various immunologic parameters in hemodial¬ ysis patients to confirm that prolonged antigenemia in these patients may be associated with depressed immunity as a result of a reduced total number of T-lymphocytes. Materials and methods

Subjects From the tissue typing laboratory, department of laboratory medicine and renal metabolic unit, department of medicine, University of Ottawa and Ottawa General Hospital Reprint requests to: Dr. D.P.S. Sengar, Department of laboratory medicine, Ottawa General Hospital, 22 Bruyere St., Ottawa, ON KIN 5C5

Serum from 86 hemodialysis patients, 105 healthy sub¬ jects "at risk" (26 artificial kidney unit [AKU] staff and 79 laboratory staff) and 160 regular hospital staff was screened for HBsAg and hepatitis B surface antibody (antiCMA JOURNAL/NOVEMBER 22, 1975/VOL. 113 945

HBs during 1973 and 1974. Hemodialysis was carried out with the Kiil parallel-flow dialyzer two to three times a week for a total of 24 to 30 h/wk in a single room with dialysis facilities for eight patients at a time. No attempt was made to isolate HBsAg-positive patients from the others. In addition, 25 renal allograft recipients were screened for 1 year after transplantation; all were receiving

immunosuppressive therapy.

HBsAg and anti-HBs determination HBsAg was determined at monthly intervals by counterimmunoelectrophoresis17 and anti-HBs by hemagglutination assay.18 Serum with a titre of 1:8 was considered positive for anti-HBs. HBsAg-coated cells were obtained from Electro-Nucleonics, Bethesda, Maryland, and anti-HBs reagent from Behring Diagnostics, Montreal. Parameters of cellular immunity Hemodialysis patients were tested for delayed cutaneous reactivity with 0.1 ml of each of the following four antigens: purified protein derivative (PPD) of tuberculin, intermediate strength (Parke-Davis); dermatophytin-O, 1:100 dilution (Hollister-Stier); two concentrations of Varidase (streptokinase-streptodornase; SK-SD), 5 U SK-2.5 U SD and 50 U SK-25 U SD (Lederle), and mumps antigen (Eli Lilly). Induration of at least 10 mm with or without erythema at 24 or 48 hours was considered a positive reaction. An in¬ dividual was considered to have the ability to manifest de¬ layed cutaneous reactivity if he showed a positive reaction to any of the four antigens tested.

Response to mitogens In vitro response of lymphocytes of 34 hemodialysis patients and 20 healthy subjects to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) was studied by a micromethod,19 in which 105 Ficoll-isopaque purified lym¬ phocytes were cultured in 0.1 ml of medium 199-Hepes, containing 33% heat-inactivated normal AB plasma, to which were added PHA and PWM. Synthesis of deoxyribonucleic acid, stimulated by the mitogens, was measured by counting the radioactivity produced by incorporated 3Hthymidine, which had been added at 48 hours.20

The healthy subjects responded to HBsAg more often by producing anti-HBs compared with the hemodialysis patients (Table II), and this difference in immune response to HBsAg was highly significant (/* 8.66; P < 0.005). In the hemodialysis patients HBS antigenemia lasted for =

more than 6 months in 82.4% (14 of 17) and for less than 1 month in only 17.6% (3 of 17). On the other hand, in all healthy individuals the duration of detectable anti¬ genemia did not exceed 1 month. In none of the six healthy subjects with transient antigenemia did anti-HBs develop during an observed period of up to 12 months. In only 4 of the 17 (23.5%) patients with HBsAg did clinical symp¬ toms of hepatitis develop, while in 11 (64.7%) the con¬ centration of serum transaminases increased transiently. In contrast, in all six of the healthy subjects with HBsAg clin¬ ical evidence of hepatitis such as nausea, vomiting, fever and jaundice developed. Anti-HBs, once formed, persisted in all the patients and in 90% of the healthy subjects over a period of 1 to 3 years. In none of the subjects with antiHBs did HBS antigenemia or clinical hepatitis develop

subsequently.

Host immune response The in vitro response of lymphocytes to PHA and PWM was normal in the hemodialysis patients (Fig. 1), which suggests that T- and B-lymphocyte function was intact in patients with HBsAg or anti-HBs. Furthermore, differential response of hemodialysis patients to HBsAg could not be explained on the basis of sex, age, time spent undergoing

hemodialysis

delayed cutaneous reactivity (Table III). obvious difference between the two groups with regard to blood transfusions: 88.2% (15 of 17) of patients in whom HBsAg was detected had previously received blood transfusions, compared with 33.3% (4 of 12) of patients in whom anti-HBs developed. HBsAg infection in renal allograft recipients In 7 of the 25 (28%) allograft recipients HBsAg became detectable 4 to 10 months after transplantation; all 7 are now asymptomatic carriers, with HBsAg titres varying from 1:256 to 1:2048. HBsAg was not detected in any of these patients during the pretransplant hemodialysis period. All seven received an average of 6 units of HBsAg-negative or

However, there

was an

Results _

Prevalence of HBsAg and anti-HBs The prevalence of HBsAg and anti-HBs in the hemo¬ dialysis patients and hospital staff is given in Table I. The combined prevalence of HBsAg and anti-HBs was higher in the staff of the AKU (57.7%) than in the hemodialysis

C3 RESPONSE

TO PHA RESPONSE TO PWM N=8 N=14

patients (33.7%).

Table I.Hepatitis B surface antigen (HBsAg) infection in 351 hemodialysis patients and hospital staff No. (and %) with HB,Ag infection No. tested With HB8Ag With anti-HBs Total 86 17(19.8) 12(14.0) 29(33.7) -

Subjects Hemodialysis patients Healthy subjects AKU staff* 26 5 (19.2) 10 (38.5) (high risk) Laboratory stafff 79 0 (0) 9 (11.4) (medium risk) Regular stafff 160 1 (0.06) 7 (4.4) (low risk) Total healthy 265 6 (2.3) 26 (9.8) ?Artificial kidney unit. tHematology, biochemistry and immunology. J Nurses and technicians other than AKU or laboratory staff.

15 (57.7) 9 (11.4) 8 (5.0) 32 (12.1)

946 CMA JOURNAL/NOVEMBER 22, 1975/VOL. 113

URAEMIC PATIENTS patients to phytohemagglutinin (PHA) and pokeweed mitogen (PWM). A = patients with hepatitis B surface antigen (HBsAg); B = patients with hepatitis B surface antibody (anti-HBs); C = patients without HBsAg or anti-HBs. NORMAL

FIG. 1.Response of uremic

blood at the time of transplantation and during the 1st month after transplantation. Five of the seven underwent hemodialysis five to seven times during the 1st month after transplantation. All patients had good renal function at 1 year, with a serum creatinine value of less than 2 mg/dl. Discussion To elucidate immunologic abnormalities in asymptomatic HB.Ag carriers Fagiolo and colleagues21 studied serum of such subjects for the presence of abnormal lymphocytes, serum immunoglobulin concentrations, and antimitochondrial, anti-smooth-muscle and anti-gamma-globulin antibodies; they found no significant differences between asymptomatic carriers (blood donors) and negative controls. However, a depressed in vitro response of purified lymphocytes to PHA has been reported by Giustino, Dudley and Sherlock22 in persistent carriers of HB.Ag. Our results indicate that uremic patients have intact Tand B-lymphocyte function as evidenced by normal in vitro response of purified lymphocytes to PHA and PWM in the presence of heat- inactivated normal AB plasma. Furthermore, there is no difference between patients with HB.Ag and patients with anti-HB, in their in vitro response to PHA and PWM or in their delayed cutaneous reactivity. We previously showed that delayed cutaneous reactivity in uremic patients was influenced primarily by the duration of uremia, for 90% of the hemodialysis patients became anergic to test antigens after 1 year of hemodialysis.20 We have recently reported that hemodialysis patients may have a normal or a higher percentage but a lower total number of T-lymphocytes than healthy controls.23 Thus, hemodialysis patients may still have depressed in vivo immunity because of a decreased total number of T-lymphocytes or a lack of certain clones, or both, in spite of having a normal in vitro response to PHA and PWM. In vivo immune response may be depressed more in patients with anemia or leukopenia, or both; such patients receive blood transfusions to correct Table Il-Immune response of patients and healthy subjects to HB,Ag infection No. (and %) with HB,Ag infection Total Subjects no. With HB,Ag With anti-HB, Hemodialysis patients 29 17(58.6) 12 (41.4)* Healthy subjects 32 6(18.8) 26 (81.2)* *Difference highly significant: x2 = 8.66; P < 0.005. Table Ill-Details of 29 hemodialysis patients with HB5Ag infection Factor Sex Male Female Age (yr) Median Range Time undergoing hemodialysis, median (days) Blood transfusions Units of blood* Median Range Cytotoxic antibody Delayed hypersensitivity Subjects positive Tests positive *For transfused patients only.

With HB8Ag (n = 17)

With anti-HB, (n = 12)

12 5

8 4

60 23-75

50 24-60

430 88.2% (15/17)

420 33.3% (4/12)

11 3-47 20.0% (3/15)

16 11-26 33.3% (4/12)

58.3% (7/12) 20.8% (10/48)

50.0% (4/8) 21.9% (7/32)

the anemia, which in turn further increases the risk of HB8Ag infection.24 We detected HB8Ag in the serum of 7 of 25 renal allograft recipients. However, it is not known whether the patients acquired HB8Ag infection from transfusions received after transplantation. Recently, Wands and colleagues25 reported an increase of up to 125-fold in the titre of HB8Ag in asymptomatic carriers in an oncology unit who were receiving antitumour chemotherapy. It is therefore possible either that our allograft recipients had HB8Ag before transplantation but that it was not detected because of the sensitivity of the technique, or that HB8Ag was not present in the circulation and appeared as a result of immunosuppressive therapy. A total of 88% of the patients with HB8Ag in this study had received blood transfusions before becoming positive for HB8Ag, compared with 33% of those with anti-HR8. These results suggest that the differential response of hemodialysis patients can probably also be attributed to the dose of antigen received or the route of immunization, or both. HB8Ag infection may spread by routes other than parenteral.2'7'24'26 If extrahematogenous infection induces production of anti-HB. more often, it may partly explain the higher prevalence of anti-HR8 in the healthy subjects at high risk in our study. It has been reported that screening of donors for HB8Ag by presently available techniques may not be adequate because transfusion of blood that is negative for HB8Ag may still induce HB8Ag-associated hepatitis in the recipients.24'27'28 Thus, the prevalence of HB8Ag in the general population is probably much higher than is realized at present. Prolonged HR8 antigenemia in hemodialysis patients may be explained on the basis of "unresponsiveness" or "tolerance", or both, induced by repeated transfusions of blood containing HR8Ag "virus" at a concentration that will not produce infection and is undetectable by presently available techniques. Once the state of "unresponsiveness" is achieved, HB8Ag "virus" may multiply and persist for long periods without harmful effects to the host. Other factors, such as genetic features, age, sex and institutionalization, have been shown to be associated with prolonged HR8 antigenemia in both healthy and sick individuals.11'29'30 A higher prevalence of HL-A3 and W19'5 and Sabell'6 antigens has been found in chronic HR8Ag carriers. A higher prevalence of HL-A3 could not be confirmed by Jeannet and Farquet16 in healthy HR8Ag carriers or by us in uremic patients.31 Our results, however, indicate that production of anti-HR8 may be negatively associated with HL-A8, and the presence of HL-A8 may therefore predispose to HR8Ag infection.31 Healthy subjects with transient antigenemia have failed to produce anti-HR8 during an observation period of 12 months. Although the reason is not clear, similar results have been reported in hemophiliacs32 (but see page 929 of this issue of the Journal). In none of the subjects with anti-HR8 has HR8 antigenemia or clinical hepatitis developed subsequently, which suggests that anti-HR8 may have some protective effect, as reported earlier.5'28 We gratefully acknowledge the assistance of Mrs. C. Keaney and Mrs. B. McDonald. This work was partly supported by Medical Research Council of Canada grant no. MA-3903. References 1. ALMEIDA JD, CHIsHoLM GD, KULATILAKE AE, et al: Possible airborne spread of serum-hepatitis virus within a haemodialysis unit. Lancet 2: 849, 1971 2. BLUMBERO BS, SUTNICK Al, LONDON WT: Australia antigen as a hepatitis virus. Am I Med 48: 1, 1970 3. Fox RA, KNIGHT AH, NIAzI 5P, et al: An epidemic of hepatitis in a renal dialysis unit. Proc R Soc Med 64: 277, 1971 4. FREIDMAN EA, THOMSON GE: Hepatitis complicating chronic haemodialysis. Lancet 2: 675, 1966 5. GOMEZ DE LA CONcHA E, EGIDO DE LOS Rios J, ORTIZ MASLIORENS F,

CMA JOURNAL/NOVEMBER 22, 1975/VOL. 113 947

et al: Immunity to hepatitis-B in a haemodialysis unit (C). Lancet 2: 461. 1.974 hazard in dialysis units. Lancet 1: 835, 1967

19. SENOAR DPS, TERASAKI P1: A semimicro mixed leukocyte culture test. Transplantation 11: 260, 1971 20. SENGAR DPS, RASHID A, HARRIS JE: In vitro cellular immunity and

7. KABOTH U, SCHORER A, KLINGE 0, et al: Endemie Australia ISH)Antigen-positiver Hepatitiden in cinem Dialysenzentrum. Dtsch Med

dialysis. mt Arch Allergy Appi Immunol 47: 829, 1974 21. FAGIoLo GB, FARINI R, CHIARAMONTE M, et al: Antibody and lymphocyte levels in HAA carriers (C). Lancet 2: 378. 1972 22. GlusriNo V, DUDLEY FJ, SHERLOCK S: Thymus-dependent lymphocyte

6. JONES P0, GOLDSMITH HJ, WRIGHT FK, et al: Viral hepatitis. A staff

Wochenschr 96: 1235, 1971

8. KUNST VAJM, BLoo JH: Australia antigen and antibody in laboratory

and other hospital personnel. Vox Sang (suppl) 24: 61, 1973 9. L.s.i M, GRIVANX C, COUROUCE-PAUTY AM: Australia antigen and

differences in 10. LONDON WT, in a chronic host response.

host response. Vox Sang 19: 359, 1970 DIFIGLIA M, SUTNICK Al, es al: An epidemic of hepatitis hemodialysis unit. Australia antigen and differences in N Engi I Med 281: 57t, 1969

11. SUTNICK Al, LONDON WT, BLUMBERO BS, es al: Australia antigen. Post-transfusion hepatitis and the chronic carrier state. Am I Dis Child 123: 392. 1972

12. TURNER GC, WHITE GBB: SH antigen in haemodialysis-associated hepatitis. Lancet 2: 121, 1969 13. WEBER KB, GROB PJ: Australia antigen and hepatitis in Hamodialysestasionen. Schweiz Med Wochenschr 102: 529, 1972 14. DUDLEY DJ, Fox RA, SHERLOCK 5: Cellular immunity and hepatitis-

associated, Australia antigen liver disease. Lancet 1: 723, 1972

15. VERMYLEN C. GOETHALS T, VAN DE PUTTE 1: Healthy carrier state and

Australia antigen liver disease (C). Lancet 1: 1119, 1972 16. JEANNET M, FARQUET JJ: HL-A antigens in asymptomatic chronic HBAg carriers (C). Lancet 2: 1383, 1974 17. CoKE DJ, HOWE C: Rapid detection of Australia antigen by counter immunoelectrophoresis. I Immunol 104: 1031, 1970 18. VYAS GN, SHULMAN NR: Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science 170: 332, 1970

in vivo delayed hypersensitivity in uremic patients maintained on hemo-

function 1972 23. SENGAR patients 24. FIELDER

in patients with hepatitis-associated antigen. Lance: 2: 850,

DPS, HYSLOP DB, RASHID A, et al: T-rosettes in hemodialysis and renal allograft recipients. Cell Inuminol (in press) H: Recipients hepatitis, an inevitable side-effect of blood

transfusion. Vox Sane 26: 368. 1974 25. WANDS JR, WALKER JA, DAvIs TT, et al: Hepatitis B in an oncology unit. N EngI J Med 291: 137t, 1975 26. WARD R, BORCHERT P, WRIGHT A, et al: Hepatitis B antigen in saliva and mouth washings. Lancet 2: 726. 1972 27. GOCKE DJ, GREENBERG HB, KAvEY NB: Correlation of Australia antigen with posttransfusion hepatitis. JAMA 212: 877, 1971) 28. OKOCKI K, MURAKANII 5, NINOMIYA K. et at: Australia antigen transfusion and hepatitis. Vox Sang 18: 289, 1970 29. SUTNICK Al, RAUNIO VK, LONDON WT, et at: New immunologic relationships of Australia antigen. Vox Sang 19: 296, 1971) 311. CAZAL P, ROBINET-LEYY M: Investigation of apparently healthy carriers of Australia antigen. Am J Dis Child 123: 383, 1972

31. SENGAR DI'S. MLEIsH WA. SUTHERLAND M, et al: Hepatitis B antigen (HBAg) infcction in an hemodialysis unit. I. HL-A8 and immune response to HBAg. Can Med Assoc 1 112: 968, 1975

32. NORDENFELI F. NILssoN IM: Antibody against Australia-antigen in hemophilia. E,.r I Clin Biol Res 16: 151. 1971

The abortion that wasn't

Last night I dreamed.., half sleeping, half waking.., of the end of your beginning.., pain, restlessness, effort.., exhaustion, then the warm wet moulding of the firm head between thighs. the statement of life in the squirming cry... and the utter joy ... peeling away the crusty layers of mothershell, till the two bodies, so recently one, became one again in the beginning of two spirits.., to nurture each other in giving gladly, till we learn to receive openly and respectfully; and you are today an almost-man, strong and lusty, like your first moments, still with unformed inner self and expecting all, but paying in unlooked-for painful joy... my last-born. PATRICIA I HARPER, MB, RS

12711 - 39th Ave. Edmonton, AB

948 CMA JOURNAL/NOVEMBER 22, 1975/VOL. 113

Hepatitis B surface antigen (HBsAg) infection in a hemodialysis unit. II. Factors affecting host immune response to HBsAg.

Serum from 86 hemodialysis patients, 105 healthy hospital staff "at risk" and 160 regular hospital staff was screened for hepatitis B surface antigen ...
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