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Post-transfusion hepatitis and hepatitis-associated antigen in New Delhi
383 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 68. N o . 5. 1974.
POST-TRANSFUSION HEPATITIS AND HEPATITIS-ASSOCIATED ANTIGEN IN NEW DELHI M. GAIHA, I. D. SHARMA, K U N A L SAHA, H. K. C H U T T A N I * AND P. N. SRIVASTAVA From Maulana A z a d Medical College, and associated Irwin and G. B. Pant Hospitals, New Delhi The present study was undertaken to assess the magnitude of the problem of post-transfusion hepatitis and its association with hepatitis-associated antigen (HAP,) and anti-HAA in New Delhi. An additional objective was to find out the carrier rates of antigen and antibody in blood donors and their relationship to abnormalities in serum transaminases.
Materials and methods Recipients
None of the 58 recipients of single or multiple blood transfusions selected for the present study had had jaundice, or blood transfusions or hepatotoxic drugs in the past, and none was found to have any evidence of liver disease clinically at the time of admission to the hospital. The first sample of blood was drawn at the time of giving the first blood transfusion in the hospital, for the purpose of estimating transaminases and of screening for HAA and anti-HAA. Subsequently the recipients were followed up once a month for 4 months, blood being taken for the tests in the outpatients' department or in their homes. I f jaundice developed, they were re-admitted to hospital. Donors 306 clinically healthy blood donors, comprising 257 professional and 49 voluntary donors, were screened for HAA and anti-HAA; 102 donors were tested for transaminases also. Controls
A control group of 1,156 subjects were screened for HAA only. Method The counter-immunoelectrophoresis method employed for the detection of HAA and anti-HAA, was a slight modification of the method used by PESENDOP,rER, ~tASSNITSKYand WEWALgA (1970). Instead of using the triple well technique, detection of antigen and antibody was carried out separately. The antibody used was obtained from a patient with haemophilia, who was given multiple transfusions.
Results Donors
Among 306 blood donors, 15 were positive for HAA alone, one for anti-HAA and one for both, giving a percentage of antigen and antibody as 5.2% and 0.65% respectively. The percentage of HAA in professional donors and voluntary donors was 5.4 and 4.1 respectively. Among 102 donors screened for transaminases also, 4 (3.9%) had minimal elevation of enzymes (mean S G O T 25 I U and S G P T 20 IU) of which only 2 were associated with antigen. We are grateful to Lt. Col. R. N. Dutta, in charge of the Armed Forces Transfusion Centre, Delhi, for his assistance with the method of detection of Australian antigen and for permission to standardize the reagents in his laboratory. Our thanks are also due to Mr. C. P. Mathur, statistician at the T.B. Centre, New Delhi, for his help in the analysis of the data. We are also thankful to Dr. R. K. Goyal, Baylor College of Medicine, Texas Medical Center, Houston, Texas, U.S.A. for a generous gift of anti-HAA antibody and standard HAA. *Requests for reprints should kindly be sent to Dr. H. K. Chuttani, Head, Department of Medicine~ Lady Harding Medical College, New Delhi.
384
POST-TRANSFUSION
HEPATITIS
AND
HEPATITIS-ASSOCIATED
ANTIGEN
IN NEW
DELHI
Recipients Among 58 recipients, 11 (18.9%) developed post-transfusion hepatitis. There were 3 icteric and 8 anicteric cases, giving a ratio of anicteric to icteric hepatitis of 2.6 : 1. Of these 11 patients with posttransfusion hepatitis 2 were associated with HAA, 2 with anti-HAA, 1 had both HAA and anti-HAA and the remaining 6 patients were negative for both. In the whole group of 58 recipients 8 (13.7%) were positive for antigen and/or antibody; 5 of these developed hepatitis while the others remained normal. This, therefore, indicates that post-transfusion hepatitis may or may not be associated with HAA and/or anti-HAA. All patients with positive tests for antigen showed hepatitis. Table I shows the time course of the appearance of HAA, anti-HAA and elevated transaminases present of the 8 patients. TABLE I. Post transfusion follow-up of 8 recipients showing HAA and anti-HAA in their sera. Age and sex
Period of transfusion in days
Appearance of HAA days a f t e r fi rs t transfusion
1
2
3
4
5
6
7
8
9
la
32F
1
6
--
37
31
--
3
Icteric
139
--
137
1
Anicteric
6
3
Anicteric
Case No.
Appearance of anti-HAA days a f t e r fir s t transfusion
Interval
Interval
between
between
4 & 6
5 & 6
Duration of HA#, and anti HAA in months
Type of hepatitis 10
6a
14M
2
--
10a
33M
3
40
--
40
12a
60M
4
--
10
--
--
--
3
No hepatitis
31a
25M
33
--
155
62
--
93
2
Anicteric
--
--
--
5
No hepatitis
2 HAA 1 anti HAA
Icteric No hepatitis
41a
40M
1
--
51a
46F
3
18
63a
45F
1
--
Range
2
Elevation of enzymes days after first transfusion
11 121
18
3
(6--40)
(2-155)
0
0
103
--
--
--
1
(18-139)
(0-31)
(93-137)
(1-5)
* ( 1 8 - 1 9 9 ) f o r all 11 c a s e s o f p o s t t r a n s f u s i o n h e p a t i t i s .
Discussion
Donors Unlike OKOCHI and MURAKAMI (1968) who found a 10 to 12-fold higher percentage of antigen in professional donors as compared to voluntary donors, in the present study we found little difference between the two groups (professional donors 5.4% and voluntary donors 4.1%). This figure is higher than those reported from America, Japan and Denmark (Table II). TABLE II. Hepatitis associated antigen in blood donors. Author
Method
Percentage of HAA positive
Place of study
1. OKOCHIet al. (1968)
Gel diffusion
1.23 (male) 0" 19 (female)
Tokyo
2.
COCKEet al. (1969)
Gel diffusion
0.73
New York
3. BANKEet al. (1971)
Gel diffusion
0.18
Denmark
4. Present study (1972)
Counter-electrophoresis
5.2
Delhi
The reason for the higher rate of antigen is not dear; it may be due to the use of the more sensitive technique of counter-electrophoresis as compared to gel diffusion used by others (GocKE and HOWE, 1970). Alternatively, it could be due to a high carrier rate of antigen in our population, as reported from South India (BLUMBERG, SUTNICKand LONDON, 1968). Previously very few workers have screened blood donors for antibody. BANKE et al. (1971) reported the percentage of anti-HAA as 0-28% as compared to 0.65% in the present study.
M. GAIHA~ L. D. SHARMA~ KUNAL SAHA, H . K. CHUTTANI AND P. N. SRIVASTAVA
385
4 of our 102 blood donors showed a minimal abnormality of transaminase level and 2 of these were associated with antigen. However, the symptoms of hepatitis were lacking in all the blood donors. OKOCI-II and MURAKAMI (1968) also reported the presence of slightly higher levels of SGPT in antigen-positive donors. GOCKEand KAVEY(1969) came across only 1 case, in which anicteric hepatitis developed a few days after donating blood. KILMAN et al. (1971) observed minimal elevation of transaminases S G O T (50 IU) and SGPT (45 IU) in ½rd of their 75 antigen-positive clinically asymptomatic donors. Recipients In the present study post-transfusion hepatitis was observed in 18.9% of the recipients. The figures reported by SHIMIZU and KITAMOTO (1963), HAMPER, PRAGERand SENIOR (1964) and SOMAYAJI, STONE and GLOVER(1967) vary from 0.1% to 64.5%. There are many possible reasons for this wide range: the criteria used for diagnosis of hepatitis, units of blood transfused, source of blood transfusion, underlying disease, and method of follow-up of the recipients. HAA was present in 3 (27.2%) of our 11 patients with hepatitis. Reported figures for the presence of HAA in post-transfusion hepatitis by OKOCHIand MURAKAMI(1968) and BLUMBERG,SUTNICKand LONDON (1968) are 13% and 34% respectively. The relatively high figures in the present study could be due to employment of counter-electrophoresis. HIRSCHMAN et al. (1969) in spite of using a less sensitive gel diffusion technique obtained a still higher rate of 74%, perhaps as a result of the weekly sampling carried out by them as compared with the monthly interval in the present study. Even higher figures than this (93%) were obtained by SHULMAN,HIRSCHMANand BARKER(1970) with the use of complement fixation technique together with weekly sampling. In the present study the time taken for the transaminases to rise in the 11 cases of post-transfusion hepatitis ranged from 18 to 199 days, while that reported by HAMPER, PRAGER and SENIOR (1964) was 45 to 105 days. The range for the appearance of HAA and anti-HAA varied from 6 to 40 days and 2 to 155 days respectively, while the antigen either preceded abnormality in transaminases or occurred simultaneously. GILES et al. (1969) reported an interval of 27 to 41 days for the appearance of antigen, which preceded a rise in transaminases by 7 to 46 days. HIRSCHMANet a]. (1969) observed a slightly longer interval of 35 to 120 days for antigen, which persisted for 3 months in the majority of cases. The duration of antigenaemia in the present study was from 1 to 5 months. The ratio of anicteric to icteric hepatitis of 2.6 : 1 in the present study is comparable to that of WALSH et al. (1970) 2-2 : 1, although KRUGMANet al. (1959) and SHIMIZUand KITAMOTO(1963) observed 12 : 1 and 10 : 1 respectively. KRUGMAN and GILES (1970) observed in one case of accidental reinfection of MS2 strain that anti-HAA was present between the 1st and 25th days, then disappeared, and reappeared from the 42nd to 139th days. In the present study it varied from 2 to 155 days. The failure of anti-HAA to prevent post-transfusion hepatitis was observed in one case in the present study; HOLLAND et al. (1969) observed this in 2 cases. However, hepatitis may have been caused by a serologically distinct agent, because sequential appearance of antibody and antigen was not demonstrated in any of the cases.
Summary A study of 58 random recipients of single or multiple blood transfusions revealed post-transfusion hepatitis in 18-9%, based upon clinical and biochemical assessment. Using counter-immunoelectrophoresis, hepatitis associated antigen (HAA) alone was detected in 27-2% of cases, HAA and/or anti-HAA was found in 45-4% cases of hepatitis, and 13.7% of all the recipients. Out of 8 patients with HAA and/or anti-HAA, 3 remained normal clinically as well as biochemically, while the remaining 5 developed either icteric or anicteric hepatitis. Of the latter, one subject was associated with both antigen and antibody, 2 with antigen and 2 with antibody alone. Among 306 asymptomatic blood donors, HAA and anti-HAA were found in 16 (5-2%) and 2 (0.65%) cases respectively, without any striking difference in the percentage of HAA in voluntary and professional donors. Of the 102 donors tested for transaminases, 4 showed a minimal abnormality in transaminases, only 2 of which were associated with the antigen. A control group of 1,156 subjects showed antigen in 2-76%. REFERENCES BANKE, O., DYBKJAER,E., NOROENFELT,E. & REINICKE, V. (1971). Lancet, 1, 860. BLUMBERG,B. S., SUTNICK,A. I. & LONDON, W. T. (1968). Bull. N . Y . Acad. Med., 44, 1566.
386
POST-TRANSFUSIONHEPATITISAND HEPATITIS-ASSOCIATEDANTIGENIN NEW DELHI
GILES, J. P., McCOLLUM, R. W., BERNDTSON,L. W., JR. & KRUGMAN,S. (1969). New Engl. J. Med., 281, 119. GOCKE, D. J. & KAVEY, N. B. (1969). Lancet, 1, 1055. , GREENBERG,H. B. & KAVEY, N. B. (1969). Ibid., 2, 248. & HOWE, C. (1970). ft. Immun., 104, 1031. HAMPER, C. L., PRAGER,n . & SENIOR, J. R. (1964). New Engl. J. Med., 271, 747. HIRSCHMAN, R. J., SHULMAN,N. R., BARKER,L. F. & SMITH, K. O. (1969). J. Am. reed. Ass., 208, 1667. HOLLA~, P. V., WALSH, J. H., MORROW, A. G. & PURCELL, R. H. (1969). Lancet, 2, 553. KLIMAN, A., REID, N. R., LILLY, C. & MORRISON, J. (1971). New Engl. J. Med., 285, 783. KRUGMAN, S. & GILES, J. P. (1970). J. Am. reed. Ass., 912, 1019. , WARD, R., GILES, J. P., BODANSKY,O. & JACOBS,A. M. (1959). New Engl. ]. Med., 261, 729. OKOCHI, K. & MURAKAMI,S. (1968). Vox Sang, 15, 374. PESENDOREER, F., KRASSNITSKY,O. & WEWALKA,F. (1970). Klin. Wschr., 48, 58. ~ , ~ &~ (1970). Bull. Wld Hlth Org., 42, 957. SHIMIZU, Y. & KITAMOTO,D. (1963). Gastroenterology, 44, 740. SHULMAN, N. R., HIRSCHMAN,R. J. & BARKER,L. F. (1970). Ann. intern. Med., 72, 257. SOMAYAJI, D. N., STONE, W. N. & GLOVER, P. B. (1967). Gut, 8, 614. WALSH, ft. H., PURCELL, R. S., MORROW, A. G., CHANOCK, R. i . & SCHMIDT, P. J. (1970). J. Am. reed. Ass., 211, 261.
POST-TRANSFUSION HEPATITIS AND HEPATITIS-ASSOCIATED ANTIGEN IN NEW DELHI M. GAIHA, I. D. SHARMA, K U N A L SAHA, H. K. C H U T T A N I * AND P. N. SRIVASTAVA From Maulana A z a d Medical College, and associated Irwin and G. B. Pant Hospitals, New Delhi The present study was undertaken to assess the magnitude of the problem of post-transfusion hepatitis and its association with hepatitis-associated antigen (HAP,) and anti-HAA in New Delhi. An additional objective was to find out the carrier rates of antigen and antibody in blood donors and their relationship to abnormalities in serum transaminases.
Materials and methods Recipients
None of the 58 recipients of single or multiple blood transfusions selected for the present study had had jaundice, or blood transfusions or hepatotoxic drugs in the past, and none was found to have any evidence of liver disease clinically at the time of admission to the hospital. The first sample of blood was drawn at the time of giving the first blood transfusion in the hospital, for the purpose of estimating transaminases and of screening for HAA and anti-HAA. Subsequently the recipients were followed up once a month for 4 months, blood being taken for the tests in the outpatients' department or in their homes. I f jaundice developed, they were re-admitted to hospital. Donors 306 clinically healthy blood donors, comprising 257 professional and 49 voluntary donors, were screened for HAA and anti-HAA; 102 donors were tested for transaminases also. Controls
A control group of 1,156 subjects were screened for HAA only. Method The counter-immunoelectrophoresis method employed for the detection of HAA and anti-HAA, was a slight modification of the method used by PESENDOP,rER, ~tASSNITSKYand WEWALgA (1970). Instead of using the triple well technique, detection of antigen and antibody was carried out separately. The antibody used was obtained from a patient with haemophilia, who was given multiple transfusions.
Results Donors
Among 306 blood donors, 15 were positive for HAA alone, one for anti-HAA and one for both, giving a percentage of antigen and antibody as 5.2% and 0.65% respectively. The percentage of HAA in professional donors and voluntary donors was 5.4 and 4.1 respectively. Among 102 donors screened for transaminases also, 4 (3.9%) had minimal elevation of enzymes (mean S G O T 25 I U and S G P T 20 IU) of which only 2 were associated with antigen. We are grateful to Lt. Col. R. N. Dutta, in charge of the Armed Forces Transfusion Centre, Delhi, for his assistance with the method of detection of Australian antigen and for permission to standardize the reagents in his laboratory. Our thanks are also due to Mr. C. P. Mathur, statistician at the T.B. Centre, New Delhi, for his help in the analysis of the data. We are also thankful to Dr. R. K. Goyal, Baylor College of Medicine, Texas Medical Center, Houston, Texas, U.S.A. for a generous gift of anti-HAA antibody and standard HAA. *Requests for reprints should kindly be sent to Dr. H. K. Chuttani, Head, Department of Medicine~ Lady Harding Medical College, New Delhi.
384
POST-TRANSFUSION
HEPATITIS
AND
HEPATITIS-ASSOCIATED
ANTIGEN
IN NEW
DELHI
Recipients Among 58 recipients, 11 (18.9%) developed post-transfusion hepatitis. There were 3 icteric and 8 anicteric cases, giving a ratio of anicteric to icteric hepatitis of 2.6 : 1. Of these 11 patients with posttransfusion hepatitis 2 were associated with HAA, 2 with anti-HAA, 1 had both HAA and anti-HAA and the remaining 6 patients were negative for both. In the whole group of 58 recipients 8 (13.7%) were positive for antigen and/or antibody; 5 of these developed hepatitis while the others remained normal. This, therefore, indicates that post-transfusion hepatitis may or may not be associated with HAA and/or anti-HAA. All patients with positive tests for antigen showed hepatitis. Table I shows the time course of the appearance of HAA, anti-HAA and elevated transaminases present of the 8 patients. TABLE I. Post transfusion follow-up of 8 recipients showing HAA and anti-HAA in their sera. Age and sex
Period of transfusion in days
Appearance of HAA days a f t e r fi rs t transfusion
1
2
3
4
5
6
7
8
9
la
32F
1
6
--
37
31
--
3
Icteric
139
--
137
1
Anicteric
6
3
Anicteric
Case No.
Appearance of anti-HAA days a f t e r fir s t transfusion
Interval
Interval
between
between
4 & 6
5 & 6
Duration of HA#, and anti HAA in months
Type of hepatitis 10
6a
14M
2
--
10a
33M
3
40
--
40
12a
60M
4
--
10
--
--
--
3
No hepatitis
31a
25M
33
--
155
62
--
93
2
Anicteric
--
--
--
5
No hepatitis
2 HAA 1 anti HAA
Icteric No hepatitis
41a
40M
1
--
51a
46F
3
18
63a
45F
1
--
Range
2
Elevation of enzymes days after first transfusion
11 121
18
3
(6--40)
(2-155)
0
0
103
--
--
--
1
(18-139)
(0-31)
(93-137)
(1-5)
* ( 1 8 - 1 9 9 ) f o r all 11 c a s e s o f p o s t t r a n s f u s i o n h e p a t i t i s .
Discussion
Donors Unlike OKOCHI and MURAKAMI (1968) who found a 10 to 12-fold higher percentage of antigen in professional donors as compared to voluntary donors, in the present study we found little difference between the two groups (professional donors 5.4% and voluntary donors 4.1%). This figure is higher than those reported from America, Japan and Denmark (Table II). TABLE II. Hepatitis associated antigen in blood donors. Author
Method
Percentage of HAA positive
Place of study
1. OKOCHIet al. (1968)
Gel diffusion
1.23 (male) 0" 19 (female)
Tokyo
2.
COCKEet al. (1969)
Gel diffusion
0.73
New York
3. BANKEet al. (1971)
Gel diffusion
0.18
Denmark
4. Present study (1972)
Counter-electrophoresis
5.2
Delhi
The reason for the higher rate of antigen is not dear; it may be due to the use of the more sensitive technique of counter-electrophoresis as compared to gel diffusion used by others (GocKE and HOWE, 1970). Alternatively, it could be due to a high carrier rate of antigen in our population, as reported from South India (BLUMBERG, SUTNICKand LONDON, 1968). Previously very few workers have screened blood donors for antibody. BANKE et al. (1971) reported the percentage of anti-HAA as 0-28% as compared to 0.65% in the present study.
M. GAIHA~ L. D. SHARMA~ KUNAL SAHA, H . K. CHUTTANI AND P. N. SRIVASTAVA
385
4 of our 102 blood donors showed a minimal abnormality of transaminase level and 2 of these were associated with antigen. However, the symptoms of hepatitis were lacking in all the blood donors. OKOCI-II and MURAKAMI (1968) also reported the presence of slightly higher levels of SGPT in antigen-positive donors. GOCKEand KAVEY(1969) came across only 1 case, in which anicteric hepatitis developed a few days after donating blood. KILMAN et al. (1971) observed minimal elevation of transaminases S G O T (50 IU) and SGPT (45 IU) in ½rd of their 75 antigen-positive clinically asymptomatic donors. Recipients In the present study post-transfusion hepatitis was observed in 18.9% of the recipients. The figures reported by SHIMIZU and KITAMOTO (1963), HAMPER, PRAGERand SENIOR (1964) and SOMAYAJI, STONE and GLOVER(1967) vary from 0.1% to 64.5%. There are many possible reasons for this wide range: the criteria used for diagnosis of hepatitis, units of blood transfused, source of blood transfusion, underlying disease, and method of follow-up of the recipients. HAA was present in 3 (27.2%) of our 11 patients with hepatitis. Reported figures for the presence of HAA in post-transfusion hepatitis by OKOCHIand MURAKAMI(1968) and BLUMBERG,SUTNICKand LONDON (1968) are 13% and 34% respectively. The relatively high figures in the present study could be due to employment of counter-electrophoresis. HIRSCHMAN et al. (1969) in spite of using a less sensitive gel diffusion technique obtained a still higher rate of 74%, perhaps as a result of the weekly sampling carried out by them as compared with the monthly interval in the present study. Even higher figures than this (93%) were obtained by SHULMAN,HIRSCHMANand BARKER(1970) with the use of complement fixation technique together with weekly sampling. In the present study the time taken for the transaminases to rise in the 11 cases of post-transfusion hepatitis ranged from 18 to 199 days, while that reported by HAMPER, PRAGER and SENIOR (1964) was 45 to 105 days. The range for the appearance of HAA and anti-HAA varied from 6 to 40 days and 2 to 155 days respectively, while the antigen either preceded abnormality in transaminases or occurred simultaneously. GILES et al. (1969) reported an interval of 27 to 41 days for the appearance of antigen, which preceded a rise in transaminases by 7 to 46 days. HIRSCHMANet a]. (1969) observed a slightly longer interval of 35 to 120 days for antigen, which persisted for 3 months in the majority of cases. The duration of antigenaemia in the present study was from 1 to 5 months. The ratio of anicteric to icteric hepatitis of 2.6 : 1 in the present study is comparable to that of WALSH et al. (1970) 2-2 : 1, although KRUGMANet al. (1959) and SHIMIZUand KITAMOTO(1963) observed 12 : 1 and 10 : 1 respectively. KRUGMAN and GILES (1970) observed in one case of accidental reinfection of MS2 strain that anti-HAA was present between the 1st and 25th days, then disappeared, and reappeared from the 42nd to 139th days. In the present study it varied from 2 to 155 days. The failure of anti-HAA to prevent post-transfusion hepatitis was observed in one case in the present study; HOLLAND et al. (1969) observed this in 2 cases. However, hepatitis may have been caused by a serologically distinct agent, because sequential appearance of antibody and antigen was not demonstrated in any of the cases.
Summary A study of 58 random recipients of single or multiple blood transfusions revealed post-transfusion hepatitis in 18-9%, based upon clinical and biochemical assessment. Using counter-immunoelectrophoresis, hepatitis associated antigen (HAA) alone was detected in 27-2% of cases, HAA and/or anti-HAA was found in 45-4% cases of hepatitis, and 13.7% of all the recipients. Out of 8 patients with HAA and/or anti-HAA, 3 remained normal clinically as well as biochemically, while the remaining 5 developed either icteric or anicteric hepatitis. Of the latter, one subject was associated with both antigen and antibody, 2 with antigen and 2 with antibody alone. Among 306 asymptomatic blood donors, HAA and anti-HAA were found in 16 (5-2%) and 2 (0.65%) cases respectively, without any striking difference in the percentage of HAA in voluntary and professional donors. Of the 102 donors tested for transaminases, 4 showed a minimal abnormality in transaminases, only 2 of which were associated with the antigen. A control group of 1,156 subjects showed antigen in 2-76%. REFERENCES BANKE, O., DYBKJAER,E., NOROENFELT,E. & REINICKE, V. (1971). Lancet, 1, 860. BLUMBERG,B. S., SUTNICK,A. I. & LONDON, W. T. (1968). Bull. N . Y . Acad. Med., 44, 1566.
386
POST-TRANSFUSIONHEPATITISAND HEPATITIS-ASSOCIATEDANTIGENIN NEW DELHI
GILES, J. P., McCOLLUM, R. W., BERNDTSON,L. W., JR. & KRUGMAN,S. (1969). New Engl. J. Med., 281, 119. GOCKE, D. J. & KAVEY, N. B. (1969). Lancet, 1, 1055. , GREENBERG,H. B. & KAVEY, N. B. (1969). Ibid., 2, 248. & HOWE, C. (1970). ft. Immun., 104, 1031. HAMPER, C. L., PRAGER,n . & SENIOR, J. R. (1964). New Engl. J. Med., 271, 747. HIRSCHMAN, R. J., SHULMAN,N. R., BARKER,L. F. & SMITH, K. O. (1969). J. Am. reed. Ass., 208, 1667. HOLLA~, P. V., WALSH, J. H., MORROW, A. G. & PURCELL, R. H. (1969). Lancet, 2, 553. KLIMAN, A., REID, N. R., LILLY, C. & MORRISON, J. (1971). New Engl. J. Med., 285, 783. KRUGMAN, S. & GILES, J. P. (1970). J. Am. reed. Ass., 912, 1019. , WARD, R., GILES, J. P., BODANSKY,O. & JACOBS,A. M. (1959). New Engl. ]. Med., 261, 729. OKOCHI, K. & MURAKAMI,S. (1968). Vox Sang, 15, 374. PESENDOREER, F., KRASSNITSKY,O. & WEWALKA,F. (1970). Klin. Wschr., 48, 58. ~ , ~ &~ (1970). Bull. Wld Hlth Org., 42, 957. SHIMIZU, Y. & KITAMOTO,D. (1963). Gastroenterology, 44, 740. SHULMAN, N. R., HIRSCHMAN,R. J. & BARKER,L. F. (1970). Ann. intern. Med., 72, 257. SOMAYAJI, D. N., STONE, W. N. & GLOVER, P. B. (1967). Gut, 8, 614. WALSH, ft. H., PURCELL, R. S., MORROW, A. G., CHANOCK, R. i . & SCHMIDT, P. J. (1970). J. Am. reed. Ass., 211, 261.