Pneumonology

P n e u m o n o l o g y 153, 109-117 (1976}

® Springer-Verlag 1976

Serologic Studies on the Occurrence of Specific Rifampicin Antibodies during Continuous Rifampicin Treatmentits Frequency and Significance Robert

Kropp,

Martin

Krfipe,

and Heinrich

Jungbluth

A b s t r a c t. From August of 1972 until February of 1974 immunologic studies on the formation of rifampicin antibodies were carried out in 171 tuberculous patients receiving rifampicin continuously in daily dosis of i 0 mg per kg body weight. In 5 of these patients rifampicin specific antibodies could be found, 4 of whom suffered from immunologic or autoaggressive disease simultaneously. The possible importance of this implication is pointed at. None of the 171 patients on continuous symptoms of rifampicin incompatibility. Key

w o r d s : Rifarnpicin

rifampicin

therapy

had clinical

- Immune-reactions.

Zusarnmenfassung. In d e r Z e i t v o n A u g u s t 1972 b i s F e b r u a r 1 9 7 4 w u r den bet 171 P a t i e n t e n a l l e r A l t e r s g r u p p e n , die i m R a h m e n e i n e r d r e i f a c h k o m b i n i e r t e n r n e d i k a m e n t S s e n T h e r a p i e R i f a m p i c i n in t~iglichen D o s e n zu 10 m g / k g K S r p e r g e w i c h t k o n t i n u i e r l i c h e r h i e l t e n , s e r o l o g i s c h e U n t e r s u c h u n gen auf d a s V o r k o m m e n y o n R i f a m p i c i n - A n t i k S r p e r n d u r c h g e f i i h r t . M e t h o disch g e s c h a h d i e s in d e r W e i s e , da2 die P a t i e n t e n s e r e n rnit O - E r y t h r o z y t e n A u f s c h w e m m u n g e n in f r i s c h e r n N o r r n a l s e r u r n in G e g e n w a r t von g e l S s t e m R i f a m p i c i n i n k u b i e r t w u r d e n , d a n a c h w u r d e an d e r d i r e k t und an d e r i n d i r e k t in C o o m b s t e s t n a c h w e i s b a r e n A g g l u t i n a t i o n o d e r N i c h t a g g l u t i n a t i o n d e r E r y t h r o z y t e n bzw. a u c h H~imolyse d a s V o r h a n d e n s e i n yon R i f a m p i c i n - A n t i k 6 r p e r n beurteilt. N u r b e t 5 d i e s e r 171 P a t i e n t e n k o n n t e n R i f a m p i c i n - A n t i k S r p e r a u f g e f u n den w e r d e n . I r g e n d w e l c h e k l i n i s c h e n E r s c h e i n u n g e n a l s A u s d r u c k e i n e r i m munpathotogisch v e r u r s a c h t e n R i f a m p i c i n - A l l e r g i e konnten w e d e r bet diesen 5 P a t i e n t e n noch bet dem tibrigen P a t i e n t e n k o l l e k t i v beobachtet werden. In d e r D i s k u s s i o n w i r d d a r a u f h i n g e w i e s e n , daf~ b e t 4 d e r p o s i t i v r e a g i e r e n d e n P a t i e n t e n n e b e n d e r T u b e r k u l o s e B e g l e i t l e i d e n b e s t a n d e n , die dern F o r r n e n k r e i s tier A u t o a g g r e s s i o n s k r a n k h e i t e n z u z u r e c h n e n sind.

110

Observations of immunologic complications during rifarnpicin treatment have called our knowledge about the side-effects of this frequently used and very effective antituberculous drug into question. Hitherto, no reliable data are available concerning the frequency and seriousness of these complications, or the possibility and frequency of subclinical immunologic reactions, i. e., without clinical manifestations. An evaluation of these problems seems necessary in order to improve the safety of rifarnpicin therapy.

PATIENTS In this study sera of 171 patients I, 121 males and 50 females (Table I), were analyzed for rifampicin antibodies. The patienis were suffering from active tuberculosis, mostly located in the lungs. Three of them were treated for tuberculosis of the kidneys, three for tuberculous lymphadenopathy, one for tuberculosis of the lungs and right tonsil. Table 2 shows the patients with extrapulrnonar tuberculosis and the incidence of other concomitant diseases. Table

age

i. Age

(years)

and

sex distribution

of 171 patients

m

f

total

6

5

ii

21 - 30

21

ii

32

31 - 40

24

6

30

41 - 5O

20

4

24

51 - 60

14

5

19

61 - 70

24

13

37

71 - 80

i0

5

15

< 20

>80

total

2

121

1

50

3

171

We express our gratitude to Dr. I. SchLitz, Berlin, and Dr. A. Poeschel, Fulda, for their friendly assistance by sending us sera of their tuberculous patients.

111

Table 2. Extrapulmonar seases

localization

diagno sis

and concomitant

Tuberculous

of kidneys lymphadenopathy

Tuberculosis

of tonsils 6

hepatosis

Chronic

alcoholism

8

Chronic

hepatitis

2

Diabetes

mellitus

7

Chronic Renal

Lupus

2

pyelonephritis insufficiency

Chronic

polyarthritis erythematodes

Bronchial Wegener'

di-

patient s

Tuberculosis

Toxic

of tuberculosis

5 (PCP)

2

visceralis

1

asthma s granulomatosis

4 1

Rifampicin was given daily in one dose of i0 mg per kg body weight on six days every week. 161 patients received the drug while under examination, having taken it for 1 month to two years before. Ten patients had stopped rifampicin treatment a few days to weeks prior to the immunologic studies. With few exceptions rifampicin was part of a combined antituberculous treatment with three drugs, mainly isoniazid, ethambutol, and rifampicin. 31 patients for a time received streptomycin, 6 patients capreomycin, 3 patients prothionamide, ii patients PAS, 4 patients pyrazinamide, and 4 patients cycloserine. Serologic

Studies

A total of 274 sera from 171 patients were tested. 136 patients were analyzed only once, 36 patients 2-6 times in intervals of some weeks. The blood samples were taken independently of complaints or clinical symptoms and drawn by usual venous puncture. After clotting the sera were won within two hours standing at room temperature, and by centrifuging and decanting. The antibody tests were done either the same day or - mostly - a few days or weeks later the sera being stored in the freezer (- 20 ° C).

112

Methods

for Testing

Native sera from the patients were brought into contact with rifampicin in the presence of erythrocytes from healthy persons of blood group 0 Rh or 0 rh suspended in their own fresh serum. This mixture was incubated at 37°C and - if hemolysis had not taken place - examined for hemagglutination in the following three ways: I. By indirect Coombs-test using polyvalent anti-human globulin sera from rabbits 2 (Behringwerke, Dade). If hemolysis was observed no further steps followed. 2. By direct hemagglutination test without changing the medium and if no agglutination was seen 3. by adding activated papain-solution (= "L(DW"-solution [see 18]) and reading for hemagglutination once more after 15 rain. Preparation

of Test

Reagents

a) Rifampicin Solution. According to the specifications of Poole et al. [27] the content of one rifampicin capsule of 150 mg produced for therapeutic purpose was dissolved as far as possible in 200 ml of 0.9% NaCI solution employing a magnetic stirring apparatus for 30 rain at room temperature This yielded an intensively red-orange colored, clear liquid with a small undissolved residuum adhering to the wall of the glassware. The clear trans. lucent solution only was used. It was prepared anew for each examination series. b) Erythrocyte Suspensions Containing Rifampicin. Following the data of G. Poole et al. ~27], W. Hasse et al. [14], and B. Kr~nig et al. [16] blood cells obtained the day before from blood samples of healthy donors were washed once with 0.9% NaCI after clotting, were resuspended in their own fresh serum, and then mixed with the rifampicin solution (0. 075 mg/ml) described above in the following manner: 0.5 ml sediment of erythrocytes, i. 0 ml own serum and I. 0 ml rifampicin solution. This suspension was diluted once more with 0.9% NaCI to a total of 12.0 ml resulting in a 4-5% cell suspension containing 0.05-0. 08 mg rifampicin/ml. A 4% suspension of the same erythrocytes without addition of rifarnpicin served as control. The

Tests

One drop of each patient' s serum was mixed with one drop of the prepared rifampicin- erythrocyte s- serum suspension (a) in test tubes for the indirect Coombs-test and (b) in the excavations of porcelain slides for the direct agglutination tests. Both mixtures, (b) in a

Only two sera reacting positively with polyvalent C o o m b s s e r u m were tested with Ig-type-specific antisera (anti-lgG, anti-lgA, and anti-IgM), showing negative results. W e did not use anti-serum against complementfactors.

113

humid chamber, were incubated in a water bath at 37°C for one hour. Then the content of the test tubes was washed three times with 0.9% NaCI and prepared for reading of agglutination by adding two drops of Coombs serum (Behringwerke, Dade) in the usual manner. If agglutinations in the sets on slides were not observed one drop of the "L0W"-solution was added, and the sets were read 15 rain later.

RE SU LT S Control sera with rifampicin antibodies from other investigators were not at our disposal. Luckily, however, the first series of analyses contained an antiserum w.ith strongly positive reaction using the indirect Coombs test. This serum served as control during all the test series. Its storing in the freezer (- 20°C) for months did not impair the positive reactivity. More than i0 of the 171 patients showed serum agglutinins to the test erythrocyte suspension. Only 5 of them proved to have specific rifampiein antibodies. The other sera contained agglutinins against blood group antigens, for instance anti-H, anti-P, and anti-C. These blood group antibodies must be payed attention to as a possible error when using human blood cells for evidence of drug antibodies. Only in 3 of the 5 positively reacting patients were the rifampicin antibodies found several times in intervals of some weeks. In the other 2 patients they could not be detected on repeated examination. In 4 patients only the indirect Coombs test was positive, in the fifth patient the papain test too, Sera presenting agglutination only with the direct tests containing no rifampicin antibodies. Four of the positively reacting patient sera showed slight hemolysis which could not be examined quantitatively. No c l i n i c a l s y m p t o m s of r i f a m p i c i n i n c o m p a t i b i l i t y could be o b s e r v e d in the 171 p a t i e n t s . T h e u s u a l l a b o r a t o r y e x a m i n a t i o n s d u r i n g a n t i t u b e r c u l o u s c h e m o t h e r a p y (SGOT, S G P T , G a m m a - G T , c r e a t i n i n e , urea} w e r e n o r m a l , e x c e p t f o r s l i g h t i n c r e a s e s of s e r u m t r a n s a m i n a s e s up to 40 mU which did not necessitate interruption of therapy and were reversible within a short time. These elevations of serum transaminases were observed mostly in patients with known hepatic damage, in particular in chronic alcoholics. The 5 patients with rifampicin antibodies are presented here in detail: I. E.B., 9, 36 years old Diagnosis: active tuberculosis of lungs, Lupus erythernatodes visceralis. During therapy with rifampicin, isoniazid, and immunosuppressive drugs, the indirect Coombs test was positive on Oct. 30th, 1972 (Monday}. After cessation of rifampiein treatment in Dec. 1972 the test proved negative on April 4th, 1973 (Tuesday}. 2. W.N.,C~, 42 years old Diagnosis: active tuberculosis of lungs. Wegener" s granulomatosis. Pulmonary embolism. Renal insufficiency. Under therapy with rifampicin, capreomycin, and ethambutol, the indirect Coombs test was positive on Sept. llth, 1972 (Monday} and on Sept. 22nd, 1972 (Friday}. 3. H.R.,(~, 40 years old Diagnosis: active tuberculosis of lungs. Bronchial asthma.

114

The therapy with rifampiein had been finished in May of 1972. The indirect Coombs test was positive on Nov. 21st, 1972 (Tuesday) and on May llth, 1973 (Friday). At that time the patient was taking isoniazid, sodium dichromoglycicum, and bronchodilator drugs. 4. L. S. ,O~, 56 years old Diagnosis: active tuberculosis of lungs. Thromboembolic disease. Hepatic steatosis. PCP. Under therapy with rifampicin, isoniazid, and ethambutol, the indirect Coombs test was positive on Sept. 20th, 1972 (Wednesday), negative on Oct. 18th, 1972 (Wednesday). 5. A.Y. ,04, 30 years old Diagnosis: active tuberculosis of lungs. Under a therapeutic regimen with rifampicin and isoniazid the indirect Coombs test was positive on Jan. 26th, 1973 (Friday) and negative on May 7th, 1973 (Tuesday).

DISCUSSION When rifampicin was introduced into the therapy of tuberculosis only hepatic toxicity was known as a possible side-effect. Meanwhile other phenomena have been disclosed as being of the utmost toxicologic interest. This include~, the capabilityofrifampicin for enzyme induction C3], the interaction with oral contraceptives [ 17, 25, 30] ~ and the possibility of a certain immunosuppressive effect [24, 26]. On the other hand Blajman et al., 1970 [2 ] and Poole et al., 1971 [ 27] published observations of thrombocytopenia, anuria, and febrile reactions in patients on intermittent high dose rifampicin treatment. These symptoms were attributed to immunopathologic phenorn ena, antibodies to rifampi¢in could be demonstrated by both authors. Subsequently, further evidence was published on patients receiving high dose intermittent rifampicin therapy [5, 7, i0, 16, 21, 22, 28] or in patients on continuous rifampicin regimen after an interruption of treatment ~i, 6, 12, 15, 19, 21, 22, 23, 28, 29]. Evidence of rifampiein antibodies was given by several authors [8, 28, 31]. In some instances such clinical complications on immunopathologic basis were observed during a daily continuous rifampicin treatment [4, II, 14, 20, 34]. These incidences due to their rareness could not be observed in our study in spite of a relatively high number of patients (n = 171). Although rifampicin antibodies have been described in intermittent rifampicin dosage without clinical complications, subclinical phenomena [ 133 may occur sometimes in high percentages. The analysis of rifampicin antibodies in vitro by means of erythrocytes in the presence of rifampicin implies the conception of a specific compound between the rifampicin-immunoglobulins and the rifampicin-(hapten)-protein complexes (albumin?) formed in protein containing medium, these soluble antigen/antibody complexes being adsorbed on the surface of erythrocytes binding complement. Patients in the Federal Republic of Germany are usually on a continuous daily rifampicin regimen - apart from special instances (for instance "intermittent" according to Schiitz [ 32] ) - which can naturally be interrupted for

115

s e v e r a l r e a s o n s , a m o n g which is i n c o r r e c t d r u g a c c e p t a n c e in u n r e l i a b l e patients. T h e r e f o r e it was n e c e s s a r y to i n v e s t i g a t e w h e t h e r r i f a m p i c i n a n t i bodies could be d e t e c t e d d u r i n g continuous r i f a m p i c i n t r e a t m e n t in a d a i l y dosage of l0 m g p e r kg body weight. T h i s s t u d y showed r i f a m p i c i n - a n t i b o d i e s by m e a n s of h e m a g g l u t i n a t i o n r e a c t i o n s ( i n d i r e c t C o o m b s t e s t s ) in 5 of 171 p a t i e n t s (= 2.9%) on c o n t i nuous rifampicin treatment, no clinical s y m p t o m s of this rifampicin-antibody formation being observed. It s e e m s worthwhile to mention that 4 of these 5 patients suffered from immunologic or autoaggressive diseases simultaneously, i.e., Lupus erylhmatodes visceralis, bronchial asthma, P C P , Wegener" s granulomatosis. Similar observations were published by Leggat (hepatic cirrhosis [20]), Esposito (M. Waldenstroem [9]) and Hasse (helminthic disease (?) [ 14]). These data raise the question whether production of rifampicin antibodies m a y be promoted or enhanced in the presence of an immunologic or autoaggressive disease. In conclusion this study seems to prove that during exact daily rifampicin treatment, immunologic reactions are to be expected only to a small extent, dangerous accidents only very rarely. The recommended laboratory controls during rifampicin treatment [ 33] seem to be sufficient provided that in addition a blood examination is done, and thrornbocytes, and serum bilirubin are checked every 4 weeks. The main attention should be directed towards avoiding therapy interruptions. The patients must be informed of this. The problem remains as to why, regarding the wide application of rifampicin, only relatively few patients suffer from severe accidents caused by immunologic reaction to the rifampicin drug. Further discussion should keep in mind our observation of concomitarit other immunologic or autoaggressive diseases in rifampicin-antibody producers and on the other hand in a broader sense the certain immunosuppressive effect - if only poor - of rifampicin.

REFERENCES

i. Blaha, H. : Symposion im Z e n t r a l k r a n k e n h a u s Gauting fiber N e b e n w i r k u n gen yon R i f a m p i c i n v o m 11.12. 1972. Zit. n a c h P r a x . P n e u m o l . 27, 434435 (1973) m 2. B l a j c h m a n , M . A . , L o w r y , R . C . , P e t t i t , J . E . , S t r a d l i n g , P. : P o t e n t i a l l y s e r i o b u s s i d e e f f e c t s of h i g h - d o s e t w i c e - w e e k l y r i f a m p i c i n . B r i t . Med. J. 1970 III, 24 3. Bolt, H. M. : N e b e n w i r k u n g e n yon R i f a m p i c i n und i h r e b i o c h e m i s c h e n G r u n d lagen. Dtsch. Med. W s c h r . 100, 63-65 (1975) 4. Bundschu, H . D . , Menz, H . P . , Hayduk, K . , S c h o m e r u s , H . , Bohle, A. : Schwere ( a l l e r g i s c h e ? ) i n t e r s t i t i e l l e N e p h r i t i s d u t c h T u b e r k u l o s t a t i k a . Verh. D e u t s c h e Ges. Inn. Med. 80, 1564-1567 (1974) 5. C a m p e s e , V . M . , M a r z u l l o , F . , Schena, F . P . , C o r a t e l l i , P. : A c u t e r e n a l f a i l u r e d u r i n g i n t e r m i t t e n t r i f a m p i c i n t h e r a p y . N e p h r o n 1_0, 256-261 (1973) 6. C o r d o n n i e r , D . , M u l l e r , 5. M. : A c u t e r e n a l f a i l u r e a f t e r r i f a m p i c i n . L a n cet 1972/II, 1364

116

7.

8. 9. i0.

ii. 12. 13.

14. 15. 16.

17. 18. 19. 20. 21.

22. 23.

24. 25.

26.

Decroix, G., Pujet, J.C., Homberg, J.C., Feldman, A., Kleinknecht, D. : Insuffisance renale aigue due ~ la rifampicine. Nouv. Presse m~d. 2, 2093-2095 (1973) Decroix, G., Sots, Ch., Pujet, J.C. : Accidents des traitements intermittents par la rifampicine. Nouv. Presse m~d. 3, 1555-1557 (1974) Esposito, R., Vitali, D. : Rifampicin and thrombocytopenia. Lancet 1972/If, 491-492 Eule, H., Iwainsky, H., K~irnbach, E., Kaluza, P., Werner, E. :M6glichkeiten einer weiteren Optimierung der Tuberkulosetherapie - Ergebnisse einer kontrollierten klinischen Studie. Z. Erkr. Aim. 141, 233-24! (1974) Ferguson, G.C. : Rifampicin and thromboeytopenia. Brit. Med. J. 1971, 5775, 638 Germann, H. J., Hoppe- Seyler, G.F., Boesken, W.H. : Akutes Nierenversagen nach Rifampicin. Dtsch. Med. Wschr. 99, 1454-1457 (1974) Graber, C.D., Jebaily, J., Galphin, R. L., Doering, E. : Light chain proteinuria and humoral immunincompetence in tuberculous patients treated with rifampin. Amer. Rev. resp. Dis. 107, 713-717 (1973) Hasse, W., Pohle, H.D., Warnecke, F., Wieck, K. :Haemolytische Krise durch Rifampicin. Prax. Pneumol. 25, 466-468 (1971) Kleinknecht, D. , Homberg, J.C. , Decroix, G. : Acute renal failure after rifampicin. Lancet !972/I, 1238-1239 KrSnig, B., Fiegel, P., Weihrauch, Th., H6ffler, D,, Jahnecke, J., Arndt-Hanser, A. : A case of severe repeated immunological reactions to intermittent rifampicin treatment. Europ. J. clin. Pharrnacol. 5, 53-57 (1972) Kropp, R. :.Rifampicin und Ovulationshemmer. Prax. Pneumol. 28, 270272 (1974) Krfipe, M..~ Die Blutgruppen des Menschen. Eine fJbersich{, S. 39. Fulda: 1969/70 Lakshminarayan, S., Sahn, S.A., Hudson, L.D. :Massive haemolysis caused by rifampicin. Brit. med. J. 1973, 5861, 282-283 Leggat, P.O. : Rifampicin and thrombocytopenia. Lancet 1971/II~ 103104 Manescu, N., Gluhorschi, G., Golea, O., Nicolcioiu, M., Schwarzkopf, A., Zosin, C. : Akutes Nierenversagen nach Rifampicin. Miinch. Med. Wschr. 116, 2161-2166 (1974) Ma±tson, K., Riska, N. : Effect of cortisone on systemic reactions provoked by rifampicin. Scand. J. resp. Dis. 53.__, 97-100 (1972) MShring, K., Asbach, H.W., Schubothe, H., Weber, S. : H~molytische Krise mit akutem Nierenversagen unter Rifampicin-Behandlung. Dtsch. Med. Wschr. 99, 1458 (1974) Nilson, B.S. : Rifampicin: an immunosuppressant? Lancet 1971/II , 374 Nocke-Finck, L., Breuer, H., Reimers, D. : Wirkung von Rifampicin auf den Menstruationszyklus und die Oestrogenausscheidung bet Einnahm~ oraler Kontrazeptiva. Dtsch. Med. Wschr. 98, 1521 (1973) Paunescu, E. : In vivo and in vitro suppression of humoral and cellular immunological response by rifampicin. Nature (London) 228, 1188 (19701

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27. Poole, G., Stradling, P., Worlledge, S. :Potentially serious side effects of high-dose twice-weekly rifampicin. Brit. Med. J. 1971/3, 343- 347 28. Pujet, J.C., Homberg, J. C., Decroix, G. : Sensitivity to rifampicin: incidence, mechanism, and prevention. Brit. Med. J. 1974/2, 5916, 415-418 29. Ramgopal, Vo, Leonard, Ch., Bhathena, D. :Acute renal failure associated with rifampicin. Lancet 1973/I, I195-i196 30. Reimers, D., Je~ek, A. : Rifampicin und andere Antituberkulotika bet gleiehzeitiger oraler Kontrazeption. Prax. Pneumol. 25, 255 (1971) 31. Schubothe, H., Seufert, C. D., Weber, S. : Chinin- und Rifampicinspezifische AntikSrper rnit h~imolytischem Effekt. Verh. Deutsche Ges. Inn. Med. 78, 905-908 (1972) 32. Schlitz, I. : Ambulante Chemotherapie der Lungentuberkulose unter besonderer Beriicksichtigung der strikt iiberwachten intermittierenden Medikamentengabe. Prax. Pneurnol. 27, 154-168 (1973) 33. Deutsches Zentralkomitee zur Bek~impfung der Tuberkulose: Merkblatt ffir die Uberwachung der ambulanten antituberk-ulSsen Chemotherapie. 2. Auflage 1970

R e c e i v e d S e p t e m b e r 4, 1975

D r . R. K r o p p B a h n h o f s t r a l ~ e 12 D-6400 Fulda