Circulating antibodies in human echinococcosis before and after surgical treatment*

Bulletin of the World Health Organization, 57 (5): 751-758 (1979) Circulating antibodies in human echinococcosis before and after surgical treatment * T. TODOROV1 & G. STOJANOV2 Antibody levels in 89 patients who had been operated on for hydatid disease were studied over a period of 4 years by complement fixation, latex agglutination, bentonite flocculation, and passive haemagglutination tests. The geometric mean titres were much higher in patients with liver echinococcosis than in those whose lungs were affected. In the first month after operation, an increase in antibody level was observed more often in the former than in the latter. The raised level usually persisted for 3-6 months. In one group of 77 patients, the serological tests either became negative 6 months to 2 years after operation or remained positive during the entire postoperative period. In the other 12 patients, who had recurrent echinococcosis, the tests did not become negative, although there was a reduction in antibody levels during the first and secondpostoperative years in 6 patients, followed by a rise when the disease recurred. Antibody levels remained high during the entire observation period in the other 6 patients. The prognosis can be consideredfavourable in patients with low pre-operative titres, or negative tests, provided the tests remain negative up to the end of the first year after operation or become negative within one and a half years. Where there is only a small reduction in antibody titre, the prognosis may still be favourable if the decrease continues to fall up to the end of the second year. Since a drop in antibody titres occurred in certain cases with recurrent infection, it is advisable to defer an opinion on the prognosis until the end of the second year. If the fall in antibody titres is followed by a steady rise, or if they remain high or show slight fluctuations, a recurrence is almost certain. The immune response in echinococcosis is the result of permanent diffusion of antigenic substances through the hydatid cyst walls. The antigenic stimu- lation induces a stable antibody level, but this stimulation is reduced or ceases when certain changes occur in the cyst wall and in the hydatid fluid, or after the surgical removal of the cyst. The antibodies, may persist in low titres for some time, but sooner or later they disappear completely (15). We have studied the nature of the echinococcus antibodies, their persistence levels, and the dynamics of the immune response during the postoperative period. We have also attempted to determine to what extent the outcome of surgical treatment could be forecast from changes in antibody titres. * This work was supported by a grant from the World Health Organization. I Lecturer in Parasitology, Department of Parasitology and Tropical Medicine, Institute of Infectious and Parasitic Diseases, Medical Academy, Sofia 31, Bulgaria. 2 Assistant, Abdominal Surgery Clinic, Institute of Gastro- enterology, Medical Academy, Sofia 27, Bulgaria. MATERIALS AND METHODS The studies were carried out on 89 patients operated on for echinococcosis. Hydatid cysts were present in the liver in 56 patients, in the lungs in 27 patients, and at other sites in 6 patients. Seventy-five of the patients were operated on for primary and 14 for secondary echinococcosis. Sera were collected 6-10 times during the first, third, sixth, and twelfth months, as well as during the second, third, and fourth year of the postoperative period, and were stored at -20°C. The patients were examined clinically several times. The complement fixation (CF) test was performed using a slightly modified version of the method previously described (14). The test volume was set at 0.5 ml (instead of 1 ml) and the reaction was carried out in the cold and read after incubating for 30 min at 37°C. The final reading was made on the next day after storage at 4°C overnight; titres of 1: 5 and 4+ were taken as positive. The latex agglutination (LA) test was performed as described by Szyfres & Kagan (12). Hytex polysty- 3850 - 751 - T. TODOROV & G. STOJANOV Table 1. Comparison of complement fixation (CF), latex agglutination (LA), bentonite flocculation (BF), and passive haemagglutination (PHA) tests in 89 patients with echinococcosis tested before operation No. of CF LA BF PHA Patients tested sera No. % o'IN. % oitested ositive positive GMTa positive positive GMT positive positive GMT positive positive GMT Total hydatid patients 89 81 91 1:28 82 92 1:92 79 89 1:73 82 92 1:8150 Primary echinococcosisb 75 67 89 1:23 68 91 1:72 65 87 1:62 68 91 1:6120 liver echinococcosis 46 41 89 1:33 42 91 1:120 41 89 1:110 44 96 1:18 000 lung echinococcosis 27 24 89 1:13 24 89 1:30 22 82 1:22 22 82 1:1 010 Secondary echinococcosis 14 14 100 1:60 14 100 1:386 14 100 1:389 14 100 1:43 350 a Geometric mean titre. b Including two patients with hydatid cysts localized elsewhere than in the liver or lung. rene latex (Hyland Laboratories) was used and a titre of 1: 5 was taken as positive. The bentonite flocculation (BF) test was based on the technique of Bozicevich et al. (2) with slight modifications. The bentonite suspension was cen- trifuged once at 500 g for 15 min and then at 750 g for a further 15 min. The test was considered positive if the titre was 1: 5 or above. The passive haemagglutination (PHA) test was performed according to the method of Garabedian et al. (6), as modified by Todorov (13). The reaction was considered positive at a minimum titre of 1: 200. Antigens were obtained from fertile hydatid fluid collected from Echinococcus granulosus cysts in sheep, and were prepared as described previously (15). The antigens were kept in small quantities at -20°C before use. RESULTS Serology of echinococcosis before operation The four serological tests were found to have almost equal sensitivities in the patients examined, the differences being not statistically significant (t< 1.96 Table 1). No differences were found in the percentage of positive tests between patients with liver and lung echinococcosis. However, the geo- metric mean titres for lung echinococcosis were much lower than those for liver echinococcosis. All patients who had been operated on for secondary echinococcosis gave positive results in all four tests and had considerably higher geometric mean titres than patients with primary echinococcosis. Serology of echinococcosis after operation The patients under study were divided into two groups, one comprising 77 patients in whom an eventual decrease in antibody levels was observed after surgery, the other comprising 12 patients in whom recurrent echinococcosis was confirmed either immunologically or surgically. First group of cases. Within four years after operation, the serological tests became negative in a significant proportion of patients. The highest per- centage of negative results was established with the CF test, followed by the BF, LA, and PHA tests in that order, the differences being not statistically significant. The tests became negative more rapidly and more often in patients with lung echinococcosis than in those with liver echinococcosis. The per- centages of patients in whom the serological tests remained positive up to the end of the observation period were: CF LA BF PHA Liver echinococcosis Lung echinococcosis Total 59 66 72 86 13 35 14 52 44 56 54 76 During the first months following operation an increase in antibody levels was observed in some 752 IMMUNOLOGY OF HUMAN ECHINOCOCCOSIS patients, this being more frequent in liver echinococ- cosis than in lung echinococcosis (Table 2). In other patients the titres increased slightly or remained unaltered, while in a smaller proportion they drop- ped immediately after operation. The rises in titre were observed as early as the first week after operation, generally reaching a maximum toward the end of the first month, and in some cases even later. The elevated antibodies persisted until the third month in 28% of the patients, up to the sixth month in 53 %, and up to 1 year after surgery in 19%; they then began to decrease. Fig. 1A shows the dynamics of the antibody response during the postoperative period in terms of the geometric mean titres. The titres, increased in all four tests during the first month; after that they began to drop. The reduction in the titres that occurred between the third month and the first year following surgery, was more pronounced in patients with liver echinococcosis (Fig. 1B) than in those with lung echinococcosis (Fig. 1C). Subsequently, the antibody titres followed a similar pattern, although they remained consistently lower in patients with lung echinococcosis. It was possible to distinguish two types of re- sponse: rapid reduction in antibody levels and delayed reduction. The rapid antibody reduction was seen in 51 cases (66%), 28 with liver and 23 with lung echinococcosis. Before operation, 59 % of these cases had low antibody titres and only 27% had high titres. After operation, an increase in antibody titres was observed in 33 % of these patients. Cases with one cyst were predominant, 22% of patients having large cysts (over 10 cm in diameter), while 21 % had multiple or secondary echinococcosis. In patients with rapid decrease in antibody, the serological tests tended to become negative usually between 6 months and 2 years after operation. This process began as early as the first postoperative month. During the first 3 months after surgery, it was mainly in the patients with lung echinococcosis that the results became negative, while in patients with liver echinococcosis it was not usually until the sixth month that negative results were obtained. Where the geometric mean titres were of the rapid reduction type, the dynamics was characterized by a steep decrease, which was most pronounced 1-3 months after operation, and by a comparatively low antibody level especially during the last 3 years of the period (Fig. 2A). The delayed type of antibody reduction was seen in 26 cases (34%), 23 with liver and 3 with lung echinococcosis. Here the antibodies persisted during the entire postoperative period. The serological tests did not become negative. Before the operation all these cases were seropositive, 8 % of patients having low titres and 92% high titres. After surgery the antibody titres were increased in 66% of the pa- Table 2. Changes in titre levels of complement fixation (CF), latex agglutination (LA), bentonite flocculation (BF), and passive hemagglutination (PHA) tests during the first postoperative month Change in titre Test and No. of Unaltered or slightlyDerad localization sera Increased increased Decreased tested No. % No. % No. % CF liver 50 27 54 17 34 6 12 lung 26 7 27 15 57 4 15 total 76 34 45 32 42 10 13 LA liver 50 27 54 17 34 6 12 lung 26 7 27 14 54 5 19 total 76 34 45 31 41 11 14 BF liver 50 25 50 18 36 7 14 lung 26 6 23 15 57 5 19 total 76 31 41 33 43 12 16 PHA liver 50 27 54 17 34 6 12 lung 26 7 27 14 54 5 19 total 76 34 45 31 41 11 14 753 T. TODOROV & G. STOJANOV Fig. 1. First group of cases: progressive reduction in geometric mean titres in the CF, LA, BF, and PHA tests in relation to the postoperative period. A: all cases; B: liver infections; C: lung infections. Fig. 3. Antibody response during the postoperative period in a patient with recurrent echinococcosis. All four tests became negative 2-6 months following the tients. Large cysts were present in 46% of patients, multiple and secondary echinococcosis in 54 %. This type was characterized by very high antibody titres (Fig. 2B), several orders of magnitude higher than those seen in the rapid type of reduction (Fig. 2A). Second group of cases. This group comprised 12 patients with recurrent hydatid disease. In 7 of them the recurrence was confirmed surgically and in 5 on Fig. 2. Postoperative changes in geometric mean titres in the CF, LA, BF, and PHA tests in patients with (A) rapid and (B) delayed type of antibody reduction. operation. An increase in the titres was observed at the end of the first year after surgery. 754 CF LA- 1:1280 A A 1:51200 1: 640 1:25600 1: 320 / 1:12800 1: 160 ,"s.1:6400 1: 80 '"'o \ 1:3200 1: 40 ; 1:1600 1: 20 \Z 1:800 , 1: 10 - 1:400 m1 5 1:200 < < 0 0, -J IL ./s. 1:102400 O 1:1280 i \ B 1:51200 01:640 / ~~~~~~~~ 1:25600 1: 320 / , 1:12800* 01:160 1:6400 :2 1 : 80 1:3200 1:1 *40 \, 4;> 1:1600 E ~~l: 20~ ~ ~ ~ ~ ~ ~~ ~1:800 E 1: 10 .- 1 :400@ o1: 5 1:200 E 0 0 0 E 0 1:160 C 1:6400 1: 80 1:3200 1: 40 /'; '' 1:1600 1: 20 ' * 1:800 1: 10lo -N 1:400 1: 5 ._ 1:200 0 -0 Before 1 3 6 12 24 36 48 operation Time after operation (months) WHaO 70409 CF - E LA --A-LA-BF 1:12800 S 1:160 ._~~\ PHA 1:64003-j1:160. - '-.cm1: 8 . ~ ;,1:3200c c .1: 40 1:1600 BL 54.L 1: 20 1:800 to~~~~~~~~~~~~~~~~:409600 ./\ B 1:04800 //\ ~~~~~~1:125600 Q:2 1:1800 8 Ei. 1: lo 1:400 ' 2 1 5 1:200 E 1 40 . ...,0600 0~~~~~~~~~~ ~~1.208002 ~~~ 1 10 ~~~~~~~~~~1:10400 2 1 5 ~~~~~~~~~~~~~1:5200 1:20 1:50 0 7 Before 1 3 6 12 24 36 48 operation Time after operation (months) CO~~~~~~~~~~~~~~~~~~~~~~~H 79 160 z 0 CF w] BF 0-pl] q 1:40St 0, A-_ 01:1600.II 0) 1:20 A,1: 800 < Y.' I * .. -% J :10 1: 400c 1:101 WO78000 CICJO (NCN )Nc CC)L r- P.- r_r- '- r- - r'-. CNO00))6 C4 ~~~~~~~~~~~~~~~~~~~WHO79487 IMMUNOLOGY OF HUMAN ECHINOCOCCOSIS the basis of immunological data. These cases had positive serology, the geometric mean titres being rather high: CF test, 1: 75; LA test, 1: 500; BF test, 1: 400; and PHA test, 1: 65 000. In 6 of these cases, the titres dropped postopera- tively but in 2 of them they rose again during the first year after surgery and in the other 4 they rose during the second year. In 1 of these patients, operated on for lung echinococcosis, the tests became negative as early as 6 months after operation, increasing again after 1 year but without attaining high values. The persistence of these titres up to the 18 months gave reason to suspect the presence of cysts. This was confirmed by X-ray examination and a second operation was performed (Fig. 3). In another patient operated on for liver echinococcosis, the titres decreased without becoming negative up to 6 months after the operation, but after about 9 months they rose again, reaching the pre-operative level. Hydatid cyst of the spleen was found surgically. In another patient with multiple liver echinococcosis, the titres dropped within 1 year of a second opera- tion being performed. They then began to rise again, attaining high values between 18 and 24 months after operation. This was undoubtedly evidence of a recurrence. In the remaining 6 cases, the high antibody titres persisted after surgery, though they fluctuated some- what. In 2 patients with proved multiple echinococ- cosis of the liver and peritoneum, particularly high levels persisted up to the end of the observation period. In 2 other patients, who had been operated on for lung echinococcosis, the antibody level re- mained high up to 4 years after surgery. On oper- ation, one of them was found to have 3 cysts in the liver (Fig. 4). In the fifth case, the antibody level showed a slight drop during the first postoperative year, but subsequently remained high for 4 years because of another cyst in the kidney. The sixth case concerned a patient operated on for liver echinococ- cosis, in whom the titres continued at a high level two years after the last operation. DISCUSSION Antibody persistence after operation There are considerable differences of opinion on the duration of antibody persistence after surgical removal of hydatid cysts. Fairley & Kellaway (5) and Sorice et al. (11) maintain that the CF test remains strongly positive for up to 9-12 months, while Abou-Daoud & Schwabe (1) claim that a positive test can be obtained for as long as 11 years. However, our studies indicate that in cases with lung hydatidosis the CF test could become negative as early as 3 months after operation. By the end of the first year, complement-fixing antibody had dis- appeared in 30% of cases. Magath (8), Lass et All four tests continued to give comparatively high titres until the second operation was performed. Fig. 4. Antibody response during the postoperative period in a patient with recurrent liver echinococcosis. CF I A _______ * '> BE---~~L 1:819200 ., ' *-z PHA-X 1:409600 1:204800 E,,.\I j1:102400 1:1280 ! .g*1:51200 :641250 LL1: 320 ~. 1:12800 ~ 1: 160 - 1:6400 > csc 1:80 'r-00" ,. 1:3200 C1 : 40 1:1600 <S.i 20 ,.J~ 1:800 oo oo cn *D co O _ NuCDw si co oo (D O CM ss~~~~~~~oi - 1: 10 *-4 0 1:400 1:5 U 1:200 0 0 0 LOl LODLODLO LO odo0d i L c6o 1:C L6 1 4c6 c6 CD C-4J'71 Lfl 1- ON~~~~~~~~WO 79490 755 T. TODOROV & G. STOJANOV al. (7), and Roy et al. (10) state that the CF test yields negative results 2 months to 1 year after operation. The CF test became negative in the first year after operation in a further 26% of our cases, and the test remained positive in 44% of cases up to the end of the observation period. Sorice et al. (11) found that the PHA test re- mained positive for at least two years, after which the titres began to drop. This view, as well as that of Roy et al. (10), who state that the test becomes negative within a year and that of Lass et al. (7) who claim that the test tends to remain positive for at least 11 years, does not apply to all cases. We have shown that the PHA titre began to fall soon after the operation and that by the end of the second year it had become negative in 20% of cases, while in 76% of cases it remained positive up to the end of the observation period. The results of the present study confirm our previous view (15) and allow some additional con- clusions to be drawn regarding factors determining how long echinococcus antibodies persist after oper- ation. The duration of antibody persistence and the antibody level were influenced to a great extent by the organ in which the hydatid cysts were located; according to Yarzabal et al. (16) the condition of the cyst may also be important. With some exceptions, the decrease in antibody response is more pro- nounced and the serological tests become negative more rapidly in lung than in liver echinococcosis. In cases with high pre-operative serological titres, in- cluding those with lung localization, the rate of antibody reduction is delayed. The tests become negative more rapidly in cases with low pre-opera- tive titres. A delayed antibody reduction is observed in patients after operation for recurrences and in those with multiple hydatidosis. Cases with a transi- tory postoperative increase on titres are charac- terized by a delayed type of antibody decrease. Obviously, it would not be possible to draw general and definite conclusions concerning the duration and level of antibody persistence after surgery without taking the above-mentioned factors into considera- tion. Criteria for forecasting the postoperative course of echinococcosis Several authors maintain that the immunological data relating to the postoperative course of echinococcosis permit the results of surgical inter- vention to be evaluated and provide a pointer to the prognosis of the disease. According to Quilici et al. (9), in the case of a favourable prognosis the serological tests should become negative after 6-12 months. However, our data indicate that this is true for only 10% of patients, using the PHA test, or for 30% of patients, using the CF test. Lass et al. (7) and Coudert et al. (4) consider that successful treatment of echinococcosis is accompanied by a significant and/or a gradual decrease in antibody titres. Zorihina et al. (17) assume that following successful surgery the LA test titre falls within a few months after operation and that the test turns negative after 1-3 years. Our studies have confirmed these general conclu- sions and yielded some additional details. According to the data we obtained, a favourable prognosis can be envisaged in the following cases: (1) if the tests are negative before the operation and remain so up to the end of the first year after surgery; (2) if the titres are low before operation, become negative afterwards, and remain negative for one and a half years and (3) if the reduction in the titres is small and the tests do not become negative, but the downward trend continues up to the end of the second year after operation. According to Sorice et al. (11), in the case of recurrences of echinococcosis, the CF test should remain positive for 2 years after operation. Recur- rent hydatid disease is assumed if 6 months (3) or 1 year (11) after surgery the level of antibodies is still high. These views, however, are not applicable to all cases. We have shown that the test may remain positive for more than 2 years and that the titres may be high throughout the first year after surgery in cases with a postoperative increase in antibodies. More plausible is the claim of Lass et al. (7) that "an unchanged and suddenly increased titre would be suggestive of recrudescence". Similarly, Zorihina & Bregadze (18) established that in recurrent echinococcosis LA test titres remain high during the entire observation period. Our study confirmed in some respects the views of earlier investigators (7, 11, 17, 18). Nevertheless, it seems necessary to define more precisely the im- munological criteria indicating the recurrence of echinococcosis. We consider recurrent infection is likely in the following cases: (1) if one and a half years after operation the titres remain high or show only slight fluctuations, without a marked drop and without a transitory rise in titres during the first month after surgery; (2) if there is an increase in the titres during the first postoperative month that might obscure the actual dynamics of the antibody re- sponse, and if the titres remain high between the first and the second year, a recurrence would be likely at 756 IMMUNOLOGY OF HUMAN ECHINOCOCCOSIS 757 the end of the second year, and (3) if the titres drop after operation and then rise steadily the possibility of a recurrence towards the end of the second year after surgery should be considered. Obviously, the final judgement regarding the outcome of the surgical treatment will depend on circumstances. For some patients it may be possible to reach a prognosis earlier than for others. In order to arrive at a prognosis, at least 4 serological examinations have to be carried out, namely at 1, 6, 12, and 24 months after surgery. RtSUMIS LES ANTICORPS CIRCULANTS DANS L'ECHINOCOCCOSE HUMAINE AVANT ET APRES TRAITEMENT CHIRURGICAL La dynamique du titre d'anticorps a ete etudiee chez 89 malades ayant subi un traitement chirurgical de la maladie hydatique, et soumis pendant 4 ans a des examens serologiques comportant des epreuves de fixation du com- plement, d'agglutination au latex, de floculation a la bentonite et d'hemagglutination indirecte. La sensibilite des diverses epreuves d'immunodiagnostic appliquees avant l'intervention chirurgicale etait analogue. La moyenne geom6trique des titres etait beaucoup plus elevee dans l'echinococcose hepatique que dans l'echi- nococcose pulmonaire. Le premier mois suivant l'ope- ration, l'augmentation des titres d'anticorps etait plus frequente dans l'echinococcose hepatique que dans l'echi- nococcose pulmonaire. Ce titre d'anticorps eleve subsistait habituellement pendant 3 a 6 mois. La reduction des titres s'effectue selon une dynamique correspondante: elle est plus forte dans l'echinococcose hepatique, les titres demeu- rant cependant toujours plus eleves en ce cas. On observe deux types de diminution du titre d'anti- corps: un type rapide et un type retarde. Chez 77 malades, on a observe le type rapide, les epreuves sero- logiques devenant negatives entre le sixieme mois et la deuxieme annee suivant l'operation, plus rapidement dans la maladie pulmonaire que dans la maladie hepatique. Le type retarde observe chez 12 malades presentant une echinococcose recurrente, entrainait une diminution du titre d'anticorps, mais sans negativation des epreuves. Chez 6 de ces malades, on a observe une baisse du titre d'anticorps au cours de la premiere et de la deuxieme annee suivant l'operation, puis une nouvelle augmentation due a une recurrence tardive. Les six autres malades ont conserve un titre eleve d'anticorps pendant toute la periode d'examen. La tendance de la dynamique du titre d'anticorps pen- dant la periode post-operatoire permet d'etablir le pronos- tic. Chez les malades presentant avant l'operation une serologie negative ou un faible titre d'anticorps, le pronos- tic sera favorable si les resultats negatifs persistent jusqu'a la fin de la premiere annee suivant l'operation, ou si les resultats deviennent negatifs dans les 18 mois suivant l'operation. Si la diminution des titres d'anticorps est plus faible, le pronostic peut etre considere comme favorable si la tendance des titres d'anticorps a la baisse est observ6e jusqu'a la fin de la seconde annee. Toutefois, une baisse des titres d'anticorps a ete observee dans certains cas de recurrence. C'est pourquoi le pronostic doit etre differe jusqu'a la fin de la deuxieme annee. 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