Immunological studies on liver disease in the Mongolian People's Republic*

Bull. Org. mond. Sant) 1974, 50, 407-411Bull. Wld Hlth Org. Immunological studies on liver disease in the Mongolian People's Republic* M. A. AKHMETELI,1 V. 1. VASIL'EVA,2 N. DORDZGOTOV,4 N. NAMDAVA,5 K. S. CERNOV,3 & N. B. 9IPACEVA 3 The sera of 445 persons, mainly patients with various liver diseases, were examined for alpha-fetoprotein (AFP) and hepatitis B antigens (HB Ag) and antibodies (HB Ab). The presence ofAFP was detected in 9 sera by agar-gelprecipitation and in 22 additional sera by the more sensitive aggregate haemagglutination technique. HB Ag proved to be present in 74 sera and HB Ab was present in 34 sera. Hepatitis B antigen was present more frequently (42%) in sera containing alpha-fetoprotein than in sera that did not contain it (14.7%). The food habits of the population investigated were also studied. The fact that liver diseases are prevalent in the Mongolian People's Republic, according to the available statistical data, led to the present immuno- logical study of persons with liver complaints. In order to trace as many such persons as possible, the records of general hospitals and of the specialized cancer control centre known as the oncological dispensary, which has a special interest in liver diseases and particularly liver cancer, were studied. PREVALENCE OF LIVER DISEASES IN MONGOLIA Liver cancer Data on the prevalence of cancerous diseases in general and primary liver cancer in particular were based on an analysis of the records of the health care centres for these diseases throughout the country. Data on the prevalence of primary liver cancer in Mongolia were also obtained by studying the compo- sition of the population under observation by the oncological dispensary and from general mortality records. * This study was carried out as part of a WHO-assisted programme. 1 Director, Division of Non-Communicable Diseases, World Health Organization, Geneva. 'Chief of Laboratory, Scientific Worker, Gamaleja In- stitute of Epidemiology and Microbiology, Moscow, USSR. ' Director, Cancer Dispensary, Ulan Bator, Mongolian People's Republic. ' Chief, Surgical Department, Cancer Dispensary, Ulan Bator, Mongolian People's Republic. Between 1961 and 1971, 5 727 patients with malig- nant neoplasms were examined in the oncological dispensary, of whom 310 (5.4%) had liver cancer which occurred predominantly in men (77.1 %) and in patients over 40 years of age (96.8 Y.). Mortality and morbidity. An analysis of the records of mortality from malignant neoplasms in 1966-71 in the Mongolian People's Republic showed that over 80% of deaths were due to cancer of the stomach, oesophagus, liver, lungs, and uterine cervix. The frequency of primary liver cancer occupied third place among men and fourth place among women. Altogether 837 deaths from liver cancer were recorded in general hospitals during that period, 530 (63.4%) among men and 306 (36.6%) among women. The findings from histological study of autopsy material and from biopsies (D. Sambuupu- rev, unpublished observations) indicated hepato- cellular carcinoma in 82% of cases. A striking feature was the uneven distribution of liver cancer morbidity in the different areas of Mongolia. The highest death rates from primary liver cancer and cancer of the other organs of the digestive tract occurred in the north-eastern parts of the country. The highest prevalence of primary liver cancer was found in Hentei aimak a, where the cumulative death rate per 10 000 inhabitants for the 5 years 1966-71 was 14.4. In this same aimak, during the same period, high death rates were recorded from aThe Mongolian People's Republic is divided into 18 aimaks or administrative units. 3205 -407- M. A. AKHMETELI ET AL. cancer of the stomach (21.6), oesophagus (10.9), intestines (1.2), and kidneys (2.2). Hubsugul, Bulgan, Selenga, Central, Eastern, Suhe-Bator, and East Gobi aimaks occupied second place with regard to the prevalence of primary liver cancer. Because of the uneven distribution of primary liver cancer, the aimaks with the highest rates (Bulgan, Selenga, and Hentei) were visited and a sample survey on the patients with chronic liver disease (chronic hepatitis, chronic cholecystitis, cirrhosis, etc.) was carried out. This type of approach may have some drawbacks from the epidemiological point of view, since these areas and the groups studied may not be completely representative. However, in this first immunological study of patients with liver diseases in the Mongolian People's Republic, effort has been concentrated on identifying the groups in which immunological manifestations were most pro- nounced. Viral hepatitis In recent years, a high rate of morbidity from hepa- titis, which was registered as infectious or type A viral hepatitis, has been recorded in Mongolia. Of the 350 patients interviewed, a considerable proportion suf- fered relapses and required repeated treatment in hos- pital. Treatment of hepatitis in the home also was not rare. The interrelation of viral hepatitis and liver cancer is a very important and controversial matter on which this investigation sought to provide some data. FOOD HABITS OF THE STUDY POPULATION The north-eastern areas of the country form a typical mountain and forest zone, inhabited mainly by the Khalkhas (Mongols proper), who are mostly engaged in animal husbandry. The chief foodstuffs are meat and milk, quite often used in the form of home-made products, which are stored for a long time. Thus, during the hot months of the year when the slaughter of cattle is sharply reduced, meat is mostly used in dried form, the so-called borets. In the summer also, long-storage dairy products are pre- pared, such as aaruul (dried curds), baslag and ezgi (acid milk cheeses), tsagantos (a sort of milk crust), and shartos (a butter made from tsagantos). In winter, milk and kumyss are often kept in frozen form. The processes of preparing and storing home- made dried meat and dried curds were studied because of a possible connexion with liver diseases. The preparation of dried meat begins in November and December with the onset of the frosts. Fresh meat is cut into strips, which are hung on lines in cold barns or placed in cloth sacks and dried in the frost in the open air throughout the winter and some of the spring months. The dried meat is used for food in the hot season and partly also in the spring and autumn months owing to the impossibility of keep- ing meat fresh at that time. To make dried curds, sour milk is compressed, cut into portions, and dried in the sun during the summer months. The consumption of cereal pro- ducts, vegetables, potatoes, and fruits is limited. MATERIALS AND RESULTS For the immunological study, 445 persons were interviewed and sera collected. The majority of these persons (340) had previously had some liver com- plaint diagnosed as chronic hepatitis, chronic cir- rhosis, or cholecystitis. However, the investigated group also included persons without any specific complaints and patients with various types of cancer, other than liver cancer. Agar-gel precipitation The sera were tested in Mongolia for alpha- fetoprotein (AFP) by means of the agar-gel precipita- tion technique. It is well known that this method is simple, inexpensive, and highly specific for primary hepatocellular cancer. Of the 445 sera, 9 proved to be positive for AFP. As may be seen from Table 1, the diagnoses of these 9 cases were as follows: cirrhosis of the liver, 1; echinococcosis of the liver, 1; chronic hepatitis, 4; suspected liver cancer, 2; and no diagnosis, 1. Aggregate haemagglutination All the sera were tested again in the Gamaleja Institute of Epidemiology and Microbiology with the much more sensitive aggregate haemagglutination technique of Olovnikov and Cvetkov (3, 4). The 9 cases that were positive by the agar-gel precipita- tion technique proved to be positive also by the aggregate haemagglutination test. It is worth noting that in every case the AFP titre was over 1 : 1 024, reflecting the high level of AFP in these patients (Table 1). Besides these 9 positive cases, 22 additional sera proved to be positive for AFP when tested by the aggregate haemagglutination technique. As can be seen (Table 2), different diagnoses were made on 408 IMMUNOLOGICAL STUDY OF LIVER DISEASE Table 1. AFP titres and diagnoses of patients whose sera were tested by agar-gel precipitation No. Serum Diagnosis AFP titre 1 Suhe-Bator 58 cirrhosis of the liver 1: 2 048 2 UB-4 chronic hepatitis 1 :4 000 000 3 UB-17 chronic hepatitis 1 :65000 4 UB-18 chronic hepatitis 1 :4096 5 UB-21 liver cancer? 1:4 000 000 6 UB-27 liver cancer? 1:524 288 7 HA-26 echinococcosis of 1 :4000 000 the liver 8 UB-29 chronic hepatitis 1 :4096 9 From Arslan none 1 :8192 Table 2. AFP titres and diagnoses of patients whose sera were positive for AFP by the aggregate haemag- glutination test but not by agar-gel precipitation No. Serum Diagnosis AFP titre 1 G-9 none 1 :64 2 UB 28 liver cancer? 1: 512 3 HBE-16 atrophic cirrhosis of the 1 :1 024 liver 4 Suhe-Bator 58 chronic hepatitis 1 :512 5 Somon 6 HA-17 cirrhosis of the liver 1 512 7 HA-7 chronic hepato- 1 :2 048 cholecystitis 8 Suhe-Bator 30 chronic hepatitis 1 : 64 9 Suhe-Bator26 , 1 :64 10 Suhe-Bator 22 , , 1:128 11 Suhe-Bator 56 cirrhosis of the liver 1: 128 Somon 12 Darhan 17 post-viral hepatitis 1 :32 13 Darhan 7 hepato-cholecystitis and 1 :32 cirrhosis of the liver 14 Darhan 5 1 :32 15 Suhe-Bator 40 cirrhosis of the liver 1 :128 16 Bulgan 6 chronic hepato- 1 :128 cholecystitis 17 Bulgan 22 hepato-cholecystitis 1 :64 18 HBE-6 cholecystitis 1:64 19 G-12 none 1:128 20 HS S 31 sm none 1 :128 21 36 NCEC none 1: 256 22 0-20 liver cancer 1 :32 Table 3. Interrelation of AFP-negative and -positive sera to the presence or absence of H B Ag and H B Ab a Not HB Ag HB Ab containing Total positive positive HB Ag or HB Ab sera containing AFP 13 1 17 31 (42.0) (3.2) (54.8) (100) sera not containing 61 33 320 414 AFP (14.7) (8.0) (77.3) (100) total 74 34 337 445 (16.6) (37.6) (75.8) (100) a Figures within parentheses are percentages. these patients, all of whom require careful clinical follow-up and repeated serum investigations for AFP. Abelev et al. (1), using the same aggregate haemag- glutination technique, showed that in viral hepatitis the appearance of AFP was of reversible nature, while in liver cancer AFP was observed to persist in the sera. Hepatitis B antigen and antibody Besides measurement of AFP, the sera were investigated for hepatitis B antigen and hepatitis B antibodies by means of counter-immunoelectro- osmophoresis. Of the 445 sera, hepatitis B antigen (HB Ag) was detected in 74 cases and hepatitis B antibody (HB Ab) in 34 cases. The interrelation of the AFP-negative and -positive sera to the presence or absence of HB Ag and HB Ab are presented in Table 3. The sera containing AFP were more frequently (42.0%) positive for HB Ag as compared with the sera not containing AFP (14.7% positive for HB Ag). DISCUSSION Our results differ from other published results to the extent that, in spite of using a not very sensitive technique, we detected HB antibodies more frequent- ly. It is also noteworthy that in our investigations the HB antibodies were detected more frequently in the sera that did not contain AFP. The association of AFP-positive sera with the higher range of HB antigenaemia has also been observed by other investigators. Prince et al. (5), using the high-voltage immunoelectro-osmophoresis technique, found HB Ag in 9 out of 10 cases of 409 410 M. A. AKHMETELI ET AL. primary biliary cirrhosis and HB antigenaemia was registered in 88 patients out of 210 with hepatoma from Senegal (42%). Vogel et al. (8) showed that 30 (67%) out of 45 patients with liver cell carcinoma in Uganda were positive for AFP and that 53 % of these were positive for HB Ag. By contrast, only 13 % of the AFP- negative cases were positive for HB Ag. Simons et al. (7), using the electro-osmodiffusion technique in a study of Singapore Chinese patients with hepatocellular carcinoma, found HB Ag in 2.6% of cases. Only with the use of the more sensitive immune adherence haemagglutination tech- nique did the detection of HB Ag in the patients with hepatocellular carcinoma rise to 35.3 %, which was more than 4 times higher than in the control group. Nishioka et al. (2) also showed a significantly higher frequency of HB Ag-positive cases in patients with hepatocellular carcinoma. On the other hand, Ruoslahti & Seppala (6) found that none of the 100 apparently healthy carriers of HB antigen had a raised level of circulating alpha- fetoprotein. Based on these findings they stated that, in contrast to their earlier report, no association between a raised serum AFP and HB Ag could be established in hepatitis. Thus, the association of HB Ag and an elevated AFP means that each of these immunological markers is associated with some type of liver condition and not directly to the other marker. Nishioka (unpublished observations) found persis- tence of HB antigenaemia in patients with hepatocel- lular carcinoma. Each of 17 patients with hepatoma, who were positive for HB antigen, was bled at intervals of at least 6 weeks. A persistence of HB antigen was registered in 15 cases. In the 2 cases where HB antigen persistence was not observed, the patients suffered from infantile hepatocellular cancer and had previously received blood transfusions during surgical operations. Although usually the appearance of HB Ag is associated with blood transfusions, in these 2 cases blood transfusion was associated with the disappearance of HB Ag from the serum of the patients with hepatocellular liver cancer. It is now relatively well established that after hepatocellular carcinoma is successfully treated or if the tumour is removed by operation, the persistence of alpha-fetoprotein in the blood ceases. It is also important to study whether the persistence of HB Ag would cease following the treatment of primary hepatocellular cancer. This would enrich our knowl- edge of the nature of both AFP and HB Ag and make their use in screening procedures more effective. RISUMI ETUDES IMMUNOLOGIQUES SUR LES AFFECTIONS HEPATIQUES EN REPUBLIQUE POPULAIRE MONGOLE On a preleve des echantillons de serum chez 445 habi- tants de la Republique populaire mongole presentant divers etats pathologiques, en majorite des affections hepatiques. La technique de precipitation en milieu gelose a decele la presence d'alpha-fmetoprot6ine (AFP) dans 9 serums; la reaction d'hemagglutination-agregation, plus sensible, a permis en outre de decouvrir 22 autres serums positifs. L'antigene de l'hepatite B(Ag HB) et I'anticorps corres- pondant (Ac HB) ont e trouves respectivement dans 74 et 34 des serums examines. Dans 42% des cas, les serums positifs pour l'AFP renfermaient l'Ag HB, alors que 14,7 % seulement des serums negatifs pour l'AFP renfer- maient cet antigene. Parmi les serums positifs pour l'AFP, on comptait 3,2% d'echantillons positifs pour l'Ac HB tandis que 8,0% des serums negatifs pour 1'AFP conte- naient I'anticorps. Les auteurs examinent les relations entre 1'hepatite virale, l'hepatite chronique et le cancer du foie. Ils expo- sent les procedes locaux de preparation et de conservation des aliments en tant que causes possibles de maladies hepatiques. REFERENCES 1. ABELEV, G. I. ET AL. Assessment of the use of highly sensitive methods of determining alpha-fetoprotein for the diagnosis of hepatocellular cancer and terato- blastoma. Bjull. eksp. Biol. Med., 71 (4): 75-81 (1971). 2. NISHIOKA, K. ET AL. Natural history of Australia antigen and hepatocellular carcinoma. In: Proceed- ings of the 3rd International Symposium of the Princess Takamatsu Cancer Research Fund, Uni- versity of Tokyo, 1973, pp. 137-146. IMMUNOLOGICAL STUDY OF LIVER DISEASE 411 3. OLOVNIKOV, A. M. Immunochemistry 4: 77-80 (1967). 4. OLOVNIKOV, A. M. & CVETKOV, V. S. Bjull. eksp. Biol. Med., 68 (12): 102-104 (1969). 5. PRINCE, A. M. ET AL. SH antigen and chronic liver disease. Lancet, 2: 717-718 (1970). 6. RUOSLAHTI, R. & SEPPALA, M. Normal and increased alpha-fetoprotein in neoplastic and non-neoplastic liver disease. Lancet, 2: 278-279 (1972). 7. SIMONS, M. J. ET AL. Australia antigen in Singapore Chinese patients with hepatocellular carcinoma and comparison groups: Influence of technique sensitivity on differential frequencies. Int. J. Cancer, 10: 320-325 (1972). 8. VOGEL, C. L. ET AL. Hepatitis associated antigen in Ugandan patients with hepatocellular carcinoma. Lancet, 2: 621 (1970).