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Immunologic aspects of BCG vaccination
The Journal of Pediatrics VOL. 36
APRIL, 1950
No. 4
Original Communications IMMUNOLOGIC A S P E C T S O F BCG VACCINATION A
STRICTLY CONTROLLED STUDY I N I N F A N T S FROI~[ TUBERCULOUS I-IousEHOLDS
SOL RoY ROSENTrlAL, M.D., ProD. CHICAGO, ILL.
I W A S invited by the American Pediatric Society to discuss BCG vaccine. This is indeed a sign of a change in our thinking. B e f o r e we can consider vaccination against tuberculosis, I should like first to establish the evidence of i m m u n i t y in tuberculosis. There are m a n y who still doubt t h a t such an i m m u n i t y exists. , Here is some of the evidence which is unequivocal. 1. Koch's P h e n o m e n o n . - - I f a normal guinea pig is inoculated intradermally with virulent tubercle bacilli, no visible reaction can be n o t e d for perhaps f r o m seven to f o u r t e e n days. A t that time a small nodule develops at the site o f inoculation, and the draining lymph nodes become enlarged. The nodule becomes necrotic, breaks down, and ulcerates. At the same time the organisms are dispersed t h r o u g h o u t the b o d y of the animal and the animal finally dies of progressive tuberculosis. Oft the other hand, if one inoculates the same dose i n t r a d e r m a l l y into an infected animal which reacts to tuberculin, one will note in from t w e n t y - f o u r to forty-eight hours an intense redness, swelling, and necrosis at the site of inoculation. This necrotic area will slough out, and if smears are t a k e n of the slough, numerous tubercle bacilli m a y be noted. The local lesion m a y go on to healing and scar formation. The draining lymph nodes will not become grossly enlarged, and culturing of these lymph nodes may be negative for tubercle bacilli; at best they would be positive only between one and three weeks after the second inoculation. (In the primarily infected animals, lymph nodes can be shown to contain tubercle bacilli a few hours after intradermal injection. ~) In my own experiments I demonstrated that there is extension from the site of inoculation in a normal animal as early as five minutes after inoculation. 2 The organisms of reinfection are localized at the site of entrance in infected animals, whereas in the noninfeeted animals tubercle bacilli spread rapidly t h r o u g h o u t the body. F r o m the Institution for Tuberculosis ]~esearch, University of Illinois College of Medicine, Chicago 1Yiunicipal Tuberculosis Sanitorium (Tuberculosis Prevention Clinic) a n d l~esearch F o u n d a t i o n . Presented a t the a n n u a l meeting of The American Pediatric Society at Atlantic City, N. J., iViay 5, 1949. 399
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2. Complete Protection of Animals by Immunization.--It has been demonstrated that one tubercle bacillus wili cause progressive tuberculosis in the guinea pig. If tubercle bacilli are applied to the intact or slightly scarified skin of a guinea pig; progressive tuberculosis may develop. However, if such a guinea pig is first inoculated with avirulent tubercle bacilli, and one waits until the tuberculin test becomes positive, and places virulent tubercle bacilli on the intact or slightly scarified skin, complete protection will be afforded. Likewise, small subcutaneous or intravenous injections of from .001 to .0000001 rag. of virulent tubercle bacilli will produce no demonstrable disease in such animals. 3 Most nonsuccessful experiments have employed relatively massive doses for reinfection, e.g., 0.1 or 1 rag., which would Correspond to many million infective doses.
F i g . 1 . - - L u n g , one a n d o n e - q u a r t e r h o u r s a f t e r i n t r a v e n o u s i n j e c t i o n of BCG. T h e sept a l cells a r e s w o l l e n a n d a r r a n g e t h e m s e l v e s a l o n g t h e i n n e r a s p e c t of t h e a l v e o l a r w a l l s . N o t e t h e fiat d a r k - s t a i n i n g n u c l e i of t h e c a p i l l a r y e n d o t h e l i u m in c o n t r a d i s t i n c t i o n . ( H e m a t o x y lin 'and e o s i n X 5 0 0 . )
3. Hyperergy and Resistance to Tuberculosis in Human Beings.---If a natural primary infection in a young adult is completely healed (as depicted by a positive tuberculin test and a negative chest film), it has been shown repeatedly that this person resists the tuberculous reinfection to a much higher degree than one who has not had the primary infection. This has been shown in one of our own studies of various institutions ill the United States ~ and has been enlarged upon by Mare Daniels in a survey of some twenty reports from many parts of the world2 He found that in more than 8,000 student nurses who were initially tuberculin positive, ]73 or 2.1 per cent of them developed tu-
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berculosis in contrast to 5.8 per cent of 4,832 nurses wnose tuberculin reactions were initially negative. His own survey in England of more than 2,500 nurses was 2 per cent versus 6 per cent. In oontrast, positive tuberculin reactions in infants and children more frequently indicate active infection, which may or may not progress. The interpretation of a positive tuberculin reaction differs sharply with age.
4. The Retieulo-Endothelial System and Immunity in Tuberculosis.--The nature of immunity to tuberculosis is not entirely understood. It is generally agreed that it is not humoral, but rather connected with the cellular responses, particularly those of the rectieulo-endothelial system. I have shown 2 that the introduction into the guinea pig of avirulent tubercle bacilli, whether by the parenteral or, to a lesser extent, by the enteral route, is followed by a swelling and proliferation of the reticulo-endothelial system, especially in the lung (Figs. 1, 2, 3, and 4). This phenomenon is manifest shortly after inoculation, pro-
F i g . 2 . - - L u n g , f o r t y - e i g h t h o u r s a f t e r i n t r a v e n o u s i n j e c t i o n of ]BCG. A t u b e r c l e c o m p o s e d of a c e n t r a l g r a n u l o e y t e a n d i n t r a a e l l u l a r t u b e r c l e bacilli a n d a p r e p o n d e r a n c e of e p i t h e l i o i d cells in t h e p e r i p h e r y . ( H e m ' a t o x y l i n a n d e o s i n XS00.)
gresses rapidly for a week or two, and then gradually recedes. The injection of large doses of tuberculin (10 rag. intracutaneously) produces a severe local reaction. The reticulo-endothelial system, especially of the lung, will swell, and a marked hyperemia accompanies the swelling (Fig. 5). The blood monocyte response trails the tissue reaction. The m/1 (monoeyte/lymphocyte) ratio is increased early and during the production of the local Koch's phenomenon (Fig. 6).
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Fig'. 3 . - - L i v e r , s i x t e e n h o u r s a f t e r i n t r a v e n o u s i n j e c t i o n of ]~CG. N o t e p r o l i f e r a t i o n of t h e K u p f f e r ceils a n d t h e g r o u p i n g of s a m e i n t o s m a l l n o d u l e s . S o m e o f t h e cells c o n t a i n e d tubercle bacilli. (Hematoxylin and eosin XS00.)
F i g . 4 . - - K i d n e y , o n e a n d o n e - q u a r t e r h o u r s a f t e r i n t r a v e n o u s i n j e c t i o n of BCG, P e r t pelvic fat showing marked perivascular histiocytic mobilization and proliferation. (Hematoxylin and eosin X500.)
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HISTORY OF DEVELOPMENT OF BCG Theobald Smith s was one of the first to show t h a t the bovine tubercle bacillus differs f r o m the h u m a n t y p e bacillus. H u m a n tubercle bacilli are less v i r u l e n t for cattle t h a n for humans, and, conversely, bovine tubercle bacilli are less v i r u l e n t for h u m a n s t h a n for cattle. W o r k i n g on this principle, Von B e h r i n g 7 used h u m a n tubercle bacilli, which he a t t e n u a t e d b y culturing and aging, to vaccinate calves. The results were most e n c o u r a g i n g in t h a t tuberculosis was prevented, b u t it was soon discovered t h a t v a c c i n a t e d cows would excrete h u m a n tubercle bacilli in t h e i r milk, a n d the vaccinations were a b r u p t l y stopped.
F i g . 5 . - - L u n g , t h r e e m o n t h s a f t e r i n t r a v e n o u s i n j e c t i o n of t~CG. M o d e r a t e h y p e r e m i ' a a n d s e p t a l cell s w e l l i n g f o l l o w i n g a K o c h ' s p h e n o m e n o n d u e to old t u b e r c u l i n . (Heraatoxylin and eosin X125.)
A t the same time Calmette and Gu6rin, who were t h e n at Lille, b e g a n w o r k i n g w i t h the idea t h a t bovine tubercle bacilli m i g h t be used to immunize humans. Their w o r k b e g a n in 1908, s using an organism which was a l r e a d y iu the l a b o r a t o r y and h a d originally been isolated f r o m the milk of a tuberculous cow. T h e y b e g a n to a t t e n u a t e this organism b y growing it on ox bile potato media, which t h e y showed r e d u c e d the virulence of tubercle bacilli. A f t e r five y e a r s of such cultivation it was n o t e d t h a t this organism, which h a d originally been capable of p r o d u c i n g progressive tuberculosis in laborat o r y animals, n o w failed to do so. W h e n it was n o t e d a f t e r m a n y more y e a r s t h a t this p r o p e r t y of avirulence was constant, i m m u n i z a t i o n of animals was
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begun, first in small l a b o r a t o r y animals, 9 then in cattle, 1~ and finally in monkeys and apes. 11 Some of the early classical experiments were done as follows : Gu6rin was a veterinarian, and since the problem of bovine tuberculosis was always acute in France, Calmette and Gu6rin concentrated their earlier work on cattle. They placed five adult tuberculous cows in one row of stalls, and in the next row behind them ten calves. Six of the calves received 20 mg. of the a t t e n u a t e d organism intravenously, and the other four served as controls. The vaccinated and control calves were placed alternately. The tuberculous cows were moved every week so t h a t contamination would be uniform. A f t e r eighteen months of the experiment, none of the six vaccinated
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l~ig. 6 . - - T h e b l o o d r e s p o n s e f o l l o w i n g t h e i n t r a v e n o u s m o n o c y t e s in t h e b l o o d i n c r e a s e d f o l l o w i n g t h e m o b i l i z a t i o n m/1 r a t i o ( m o n o c y t e / l y m p h o c y t e ) increased during Koch's tuberculin.
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injection of BCG. The total of t h e t i s s u e h i s t i o c y t e s . The phenomenon p r o d u c e d b y old
cows r e a c t e d to tuberculin, whereas three of the f o u r controls were infected.
After thirty-two months, autopsy of all the animals revealed that three of the four controls presented extensive tuberculous lesions in the lungs and lymph nodes, only one of the vaccinated showed a small lesion in the lung and lymph nodes, while the others showed no lesions whatsoever. A similar experiment was done on monkeys. Unfortunately, the experiments were only partly successful since so many of the animals were tuberculous when they arrived in Europe. The project was moved to French Guinea, the natural habitat of the monkeys, and the same type of experiment was done. Tuberculous animals, vaccinated animals, and nonvaccinated animals were
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housed in the same cage. Practically all of the control monkeys and chimpanzees died os tuberculosis, whereas none of the vaccinated animals showed signs of the disease. 11 After numerous similar experiments it was definitely established that the attenuated organism did not produce progressive disease in animals, and that it would protect against natural infection by virulent tubercle bacilli. Such was the beginning of the bacillus Calmette and Gu6rin (BCG). THE BACILLUS
Now that I have discussed some of the immunological properties of the organism, I wish to discuss some of the properties of the bacillus itself. It behaves much the same as a bovine tubercle bacillus on culture. It takes about a week or ten days to grow on egg media, and produces coiled colonies which are butter yellow in color. If special methods are used, however, it is very simple to dissociate this organism into a smooth (Fig. 7) and a rough form (Fig. 8). The accompanying figures will demonstrate the difference in morphology.
Fig.
7.
Fig.
8,
Fig. 7 . - - S m o o t h " S " B C G colonies. N o t e e d g e s s l o p i n g into m e d i u m ( D E 7.2). A g e of c u l t u r e , 20 d a y s . Source, o r g a n s of i n f e c t e d ' a n i m a l s . F i g . 8 . - - R o u g h "1~" B C G colonies on L b w e n s t e i n ' s m a l a c h i t e g r e e n e g g m e d i u m ( p H 6.1). T h i s is t h e t w e l f t h g e n e r a t i o n f r o m a n o r i g i n a l blood c u l t u r e . A g e of colony, 5 d a y s . N o t e t h e s i n g l e l a r g e g r a n u l a r colonies, t h e coiled a n d t h e c r a t e r l i k e o n e s c o m p o s e d of s i m i l a r g r a n u l e s , T h e d e m a r c a t i o n of c o l o n y a n d m e d i u m is d i s t i n c t .
Neither type of colony will produce progressive tuberculosis if inoculated into guinea pigs. We have inoculated thousands of animals, and we have never seen progressive tuberculosis following BCG inoculation. Some of the early work done by Petroff 1~ and others, 13 in which they claimed that the dissociate strains were virulent, has not been repeatable since then. The morphology of the organism is of special interest. It is an acid-fast curved rod much the same as the bovine tubercle bacillus (Fig. 9). Using special methods, including the electron microscope, shadow-casting, and hyper-
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tonic solutions, certain characteristics of the o r g a n i s m have been disclosed which are of p a r t i c u l a r interest. I found (with Dr. C. I. Reed and B. P. Reed) that the bacillus has a definite capsule (Fig. 10), and granules can be f o u n d (Fig. 11) which are, most likely, the origin of new bacilli. BCG is an a v i r u l e n t bovine tubercle bacillus which retains the cultural and morphologic characteristics of bovine m y c o b a c t e r i u m .
Fig.
9,--Bacillus Calmette-Gudrin showing smooth surface and pol'ar bodies. Electronmicro~ scopic photo following siladow casting with chromium. (X45,500.)
F i g . l O . - - B a e i l l u s C a t m e t t e - ~ u d r i n a f t e r s h r i n k i n g in h y p e r t o n i c s o l u t i o n a n d s h a d o w Note definite capsule und shadow cast by irregular surface. (X45,500.)
casting.
VACCINATION
Vaccination in Humans.--After n u m e r o u s studies in animals, and a f t e r showing the avirulence of the organism, Calmette and Gu6rin were still rel u c t a n t to use it in humans. I t was Well-Hall614 who was allowed b y Professor Calmette to use the vaccine first in h u m a n s ; this was e a r l y in 1921, a f t e r there h a d been 230 consecutive t r a n s f e r s f r o m the original s t r a i n over a period of t h i r t e e n years. Methods of Vaccination.--The question arose at once as to the best method of v a c c i n a t i n g humans. Since B e s r e d k a ' s influence was s t r o n g at this time, it was decided to use the oral route. I t was soon learned, however, t h a t the
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rate of conversion of the tuberculin reaction following this method of vaccination was very low; if ]0 rag. of BCG were given orally on the third, fifth, and seventh day of life, a positive yon Pirquet's test occurred in only ]5 per cent of the infants after three months. More recently in Brazil, Arlindo de Assis has been using massive doses of vaccine by mouth. ~G He has given as much as 90 rag. in the first 10 days of life, and has reported 83 per cent positive reactors after the fourth month (though he used 10 rag. of 0. T. as a test dose).
Fig.
ll.--Bacillus
Calmette-Gudrin.
Same treatment (•
as
in F i g .
10.
N o t e definite g r a n u l e s .
Weill-Hall6,17 using the subcutaneous method, obtained 100 per cent conversion after nine weeks by the administration of from .02 to .04 rag. of vaccine. Unfortunately, there were many complications, including cold abscesses which persisted for long periods of time. Wallgren TM later suggested and used the intradermal route, and this method produced a high degree of conversion; superficial ulcers occurred in a high percentage of the cases, however, and if the vaccine was not administered properly, small abscesses occasionally resuited. One must be extremely careful with this method in order to avoid complications. Wa]lgren, using 0.1 rag., noted 100 per cent positive reactors after seventeen weeks. In an effort to avoid complications, I described the scarification method of multiple punctures in 1937. 29 The principle of this method was the introduction of minute doses of vaccine at multiple sites into the superficial layers
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of the skin transcutaneously, r a t h e r than a single large dose intradermally, which so often leads to necrosis and slough. In the original procedure a drop of vaccine was placed on the skin, and t h i r t y punctures were made with a sewing-type needle through the vaccine into the skin over an area 3 • 11/2 cm. square.
2'ig. 1 2 . - - F a p u l e s
t e n d a y s following" v a c c i n a t i o n in a young" a d u l t .
Note
discreteness.
R e c e n t l y we have developed a stainless steel disk 1 inch in diameter in which there are thirty-six prong's and which can be controlled by a small magnet. 2~ The entire vaccination is accomplished with one pressure. A week or so a f t e r this m e t h o d is used, small 2 to 3 ram. papules develop (Fig. 12), remain s t a t i o n a r y for f r o m two to three weeks, then regress and heal without gross evidence of scarring. I n infants the lesions are minute, about 1 ram. in diameter, and so/netimes difficult to see; in fact, the m o t h e r r a r e l y knows on which arm the child was vaccinated. The draining l y m p h nodes m a y become slightly enlarged, and m a y be p a l p a t e d if a special effort is made. T h e y never enlarge to become grossly visible, and never b r e a k down. The original method is modified in F r a n c e 21 b y making numerous scratches t h r o u g h a drop of vaccine, and in New York b y the spring-actuated multiple p u n c t u r e a p p a r a t u s of Birkhaug. 22
Technique of Using Multiple Puncture Disk.--Cleanse the skin over the deltoid region of the left arm with acetone and allow it to d r y thoroughly. Place a drop of vaccine on the arm with a capillary tube or from an ampule (Fig. 13). Grasp the disk with the alcohol-cleansed metal plate of the magnet, allowing the wide margin of the d~sk to e x t e n d b e y o n d the m a g n e t and away f r o m the operator. It should be held at a 30 degree angle. Tap the drop of
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vaccine with the wide margin of the disk, and pull it along so that an area 2.5 cm. in diameter is covered with vaccine. Dip the points of the disk into the vaccine, rotating the disk slightly so that the points become moistened.
Fig. 13.--The "set up" for vaccinating.
Note saw, capillary tube with vaccine, disk, and magnet.
l~ig. 14.--~r of u s i n g m u l t i p l e p u n c t u r e d i s k . N o t e p o s i t i o n - - h o l d e r in p a l m J u s t beneath the index finger, and the other hand grasping the arm underneath, and tensing skin. T h e d i s k is h e l d in p l a c e b y m a g n e t i c p l a t e .
Place the disk in the center of of the vaeeine and move the metal plate of the magnet so that it is now centered on the disk; This will avoid bending the metal. With the butt of the magnet in the palm, just beneath the index finger, and with the other hand grasping the arm underneath and tensing the skin appreciably (Fig. 14), apply pressure so that the points of the disk are well buried in the skin. In adults considerable pressure is used, whereas in newly born and older infants only enough pressure is applied so that the penetration of the points is readily felt through the palm of the operator's hand. With the pressure still exerted as above, rock the magnet and disk forward and backward and from side to side several times. Be sure to maintain the original pressure. At this point release the grasp on arm and slide the
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magnet away, off the disk, and toward the operator, maintaining the pressure, though slightly less than originally.' Following a successful procedure the disk remains on the arm, lying flat, with the points still buried in the skin. If the points are on top of the skin, then the procedure must be repeated. With the thumb and index finger, grasp the disk at the edge opposite the wide border and gently lift it away from the skin. There should be a feeling of the points disengaging themselves from the skin. Tap the vaccine again with the wide margin of the disk so that each perforation of the skin is covered with vaccine.
F i g . 1 5 A . - - S i t e of i n j e c t i o n f o l l o w i n g m u l t i p l e p u n c t u r e t e c h n i q u e . Note minute lesion just beneath epithelium and 'absence of dilated polymorphonuclear-fllled lymphatics. (Guinea p i g s k i n , five h o u r s a f t e r i n j e c t i o n . )
Do not use much pressure, since it will press the vaccine out of the perforations. Allow the vaccine to dry on the arm without dressing. Do not wash the arm for twenty-four hours. No other precautions are necessary. The disks are inexpensive and may easily be sterilized by boiling. The microscopic lesions produced by the multiple puncture method are of interest in that the vaccine enters into the most superficial layers of the skin, usually just beneath the epidermis. Since only small doses are delivered at any one point, there is never any abscess formation. Because the vascular
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supply at this level in the skin is relatively great and the ceils of the retieuloendothelial system are most numerous, many tubercle and giant cells form (Figs. 15, A and B).
Fig. 1 5 B . - - N u m e r o u s g i a n t cells and histiocytie proliferation, ten days a f t e r injection.
In contrast, it is ahnost impossible to inject material by intradermal injection as superficially as by the transcutaneous method, and the vaccine is introduced by that method into the portion of the skin which is poor in blood supply and reticulo-endothelial cells. Because of the relatively large dose at one point a small area of necrosis forms which is surrounded by a cuff of pus cells which partially occludes the lymphatics and further favors central necrosis (Figs. 16, A and B). Since this necrotic material must either be extruded or absorbed, ulcers form unless absorption takes place. The Rapidity of Development of Tuberculin Reactions.~It is possible to produce a positive tuberculin test in guinea pigs in from one to two weeks following a single puncture through a drop of vaccineY a In newborn infants,
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8. F i g . 1 6 . - - A , Site of i n j e c t i o n f o l l o w i n g c a r e f u l s u p e r f i c i a l i n t r a d e r m a l inoculatiorf ( g u i n e a p i g ) . N o t e t h a t c e n t e r of l e s i o n is d e e p in t h e r e t i c u l u m l a y e r 'and n e c r o t i c ( 1 ) ; a n d a b r o a d zone of p o l y m o r p h o n u e l e a r i n f i l t r a t i o n s~w~ouncls site ( 2 ) . (l~our h o u r s a f t e r i n j e c t i o n h e m a toxylin a n d eosln xSO.) 13, I/]dge of lesion showing" in (A). Note that the Polymorphonuclear cells are in dilated lymphatics. (l-Iematoxylin a n d eosin XI$O.)
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children, and adults we have tried f r o m t h i r t y to n i n e t y punctures, and v a r y ing concentrations of vaccine, but at the p r e s e n t time we employ the multiple p u n c t u r e disk and vaccine which contains 15 mg./ml, moist weight of organisms. Positive tuberculin reactions a p p e a r as early as eleven to thirteen days following vaccination in infants, a n d they are almost u n i v e r s a l l y positive at one month. W e m a y get as high as 90 per cent conversion in older child r e n and adults in two w e e k s ' time. The complications following this method are p r a c t i c a l l y nil.
Immunity Following ]Taccination.--I will not go into the literature deeply in discussing the i m m u n i t y c o n f e r r e d b y BGG vaccination. All of y o u are f a m i l i a r with the excellent w o r k of the Scandinavians, 24 the French, 25 the Americans, ~6 and a host of others all over the world. Up to the p r e s e n t time there have been almost 40 million vaccinations p e r f o r m e d , 30 million in J a p a n alone. 27 The n u m b e r done in Russia is not known, but it would p r o b a b l y g r e a t l y enlarge these figures. There is u n a n i m i t y in p r a c t i c a l l y every r e p o r t t h a t a definite r e d u c t i o n in the m o r b i d i t y and m o r t a l i t y of tuberculosis follows B C G vaccination. M a n y of the reports have been criticized because of the lack of controls. W e have t r i e d to set up e x p e r i m e n t s in Chicago which would meet all the criticisms concerning contro]s which h a v e been m a d e in the past. as I n this s t u d y our control group subjects have been selected in as u n b i a s e d a m a n n e r as we t h i n k possible. These infants were exclusively f r o m tuberculous tim> ilies. T h e y were r e f e r r e d to us b y the Chicago municipal tuberculosis clinics and b y p r i v a t e physicians a n d clinics. The diagnostic d a t a and the results of the s p u t u m e x a m i n a t i o n of the tuberculous contact were submitted, and all x-rays were reviewed b y our clinic before the diagnosis was accepted. W i t h the b i r t h of the child, i m m e d i a t e s e p a r a t i o n was a r r a n g e d if the m o t h e r was t u b e r c u l o u s ; if other m e m b e r s of the f a m i l y were tuberculous, the child was allowed to s t a y in the hospital as long as the m o t h e r was there. ]n e v e r y ease the child was t h e n placed in one of our own foster homes. Visits to the foster home were m a d e b y our p e d i a t r i c i a n e v e r y week. The vaccin,~tion or placebo was done in the foster home. The d u r a t i o n of isolation r a n g e d ft'om six weeks to four and one-half months, d e p e n d i n g u p o n the t y p e of ease. W h e r e the m o t h e r had f a r a d v a n c e d disease and a positive sputum, for example, the child was not v a c c i n a t e d until three months a f t e r birth, p r o v i d i n g there was a n e g a t i v e x - r a y and a negative tuberculirt test. W h e n it was the f a t h e r who was tuberculous, the child was vaccinated three to seven days after birth. I n f a n t s were usually r e t u r n e d to their homes six weeks a f t e r vaccination, p r o v i d e d the M a n t o u x test had become positive in v a c c i n a t e d individuals, or' if it was n e g a t i v e in children who were controls and there was no open source ease. E i g h t y per cent were r e t u r n e d in this manner. The division of eases was done b y a m a s t e r chart. The doctor who did the v a c c i n a t i n g n e v e r m a d e the choice as to w h e t h e r the child was to be a contr'o] or vaccinated. The eases were strictly a l t e r n a t e d in categories d e t e r m i n e d b y
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the types of source cases. Children w h o were to return to a tuberculous env i r o n m e n t were v a c c i n a t e d w i t h twice the dose used for the n o n c o n t a c t infants (i.e., t w o series of thirty punctures with the straight needle, or, more recently, a series of two disk punctures). Progress of the infants w a s f o l l o w e d by means of clinic visits at approximately six-month intervals; t h e y were theu tuberculin tested, x-rayed, and the a m o u n t of contact w h i c h they had had during the preceding period w a s ascertained. B e t w e e n the clinic visits the infants were seen at intervals by a nurse in the home. It m a y be seen in the table that there were 139 infants in the vaccinated group and _128 in the control group. There were practically the same number of v a c c i n a t e d and control group infants in each c a t e g o r y (Table I), according to the type of tuberculosis in the source case. The a m o u n t of actual contact was similar, tho.ugh the vaccinated group had the more contact (186 person years contact with the mother in the vaccinated group as against _160 in the controls) (Table II). TABLE I .
~OURCE OF ~ONTACT AND TYPE OF TUBEI~CULOSIS AT ]~IRTH OF ~HII,D
MINIMAL
ATED
CO~TROL
11
]1
43
10
9
18
VACCIN-
CONTACT SOURCE
Mother (Alone or with others) Father (Alone or with others)
MODEgATELY ADVANCED VACCIN- GONATED TROL
POSITIVE SPUTU~
FAg ADVANCED VAGCINATED
CONTgOL
VACCINATED
CONTROL
41
33
36
27
24
17
17
14
14
14
F o l l o w i n g v a c c i n a t i o n there was -100 per cent tuberculin conversion, and this remained constant for almost five and one-half years (Table III). In the control group the number of individuals at each age was practically the same as for the vaccinated. F o r example, at birth there were 131 and 128, respectively; at ]9 to 24 months, there were 102 and 100; and at 61 to 66 months, there were 40 and 40. The degree of exposure is evident w h e n it is noted that at 61 to 66 months, 57.6 per cent of the n o n v a c c i n a t e d control group infants reacted to tuberculin ( V o l l m e r patch test) (Table I V ) . The a m o u n t of contact must have been great since in our so-called n o n c o n t a c t group only 32.9 per cent of the infants had a positive reaction at that age, and this n o n c o n t a c t group of infants lived in the most h e a v i l y infected district of Chicago? s TABLE ]-I. AMOUNT OF CONTACT W I T t I :PULMONARY TUBEI~CULOSIS VACCINATED
Continual~ w i t h mother Average Continual~ w i t h father Average Continual~ w i t h others Average Intermittent, w i t h any Average
186.5 2.8 72.2 2.8 11.9 1.3 37.3 2.2
person-years years person-years years person-years years person-years years
I
CONTROLS
160.1 2.6 73.1 3.3 25.1 2.5
person-years years person-years years person-years years 56.2 person-years 2.4 years
ROSENTEAL: TABLE
III.
I1K1Vs
TUBERCULIN
I~EACTIONS
]FOLLOWING A SINGLE GROUP OF INFANTS
AGE (]Y[O.)
NO. REGISTERED
NO. TESTED
0-3 4-7 8-12 13-18 19-24 25-30 31-36 37 42 43-48 49-54 55-60 61-66
131 123 I18 111 102 91 78 72 63 ~4 48 40
124 116 107 95 87 77 69 59 52 43 36 27
TABLE
IV.
AGE
(MO.)
0-3 4-7 8~12 13-18 19-24 25-30 31-36 37-42 43-48 49-54 55-60 61-66
TUBERCULIN
415
ASPECTS OF BCG VACCINATION
ID~EACTIONS IN THE NO.
NO. POSITIVE
124 116 107 95 87 77 68 58 51 42 35 26
CONTACT
CONTROL
NO.
REGISTERED
TESTED
128 124 115 109 100 93 86 79 73 66 52 40
128 120 110 100 91 83 75 70 64 53 43 33
IN THE
VACCINATION
CONTACT
% POSITIVE
100 100 100 ]00 100 100 98.6 98.3 98.1 97.7 97.2 96.3
GROUP
OF INFANTS
NO.
v/o
POSITIVE
POSITIVE
0 9 20 41 45 46 42 41 36 31 24 19
0 7.5 18.2 41 49.5 55.4 56 58.6 56.3 58,5 55.8 57.0
There were twice the n u m b e r of cases of tuberculosis in children in the control group as a m o n g the v a c c i n a t e d (six cases against three) (Table V). W h a t is more significant is the f a c t t h a t only one child f r o m the vaeci~lated group needed hospitalization, while four f r o m the control group required it! Indeed, in the v a c c i n a t e d group, two of the children were diagnosed as tuber' eulous only because of calcified lesions in the lung; the t h i r d showed a small area of infiltration in the left hilus which calcified in a short period of time. TABLE V. INCIDENCE OF TUBERCULOSIS IN THE CONTACT GROUP OF CHILDREN
Total number of children Person-years observed Cases of tuberculosis Total number Rate "x Children hospitalized Total number :Rate ~ D e a t h s from t u b e r c u l o s i s Total number R a t e "X" Deaths from other causes Total number :Rate ~ *Rate per t h o u s a n d p e r s o n - y e a r s observation,
VACCINATED 131 459 3 6.5
i
CONTROLS 128 459 6 13
1 2.2
4 8.7
0 0
3 6.5
2 4.3
2 4,3
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On the other hand, t h r e e of the six children in the control group died. All were autopsied and were f o u n d to h a v e a t y p i c a l p r i m a r y lesion in the lung w i t h s p r e a d to tile hilus l y m p h nodes; in one ease there was a m i l i a r y tuberculosis and tuberculous meningitis. These f e a t u r e s are most striking, sinee it a p p e a r s t h a t BCG Vaccination m a y p r e v e n t v i r u l e n t tubercle bacilli f r o m establishing themselves in the host, or if the infecting dose is large and they do get a foothold, their s p r e a d f r o m the site of entrance is inhibited. I t is m u c h the same as I have described in e x p e r i m e n t a l animals.
Fig. 17.~D. F., a w h i t e f e m a l e c h i l d v a c c i n a t e d a t 5 w e e k s o f a g e . X-ray taken at 5 years of age. Mother and fa.ther both tuberculous. Continuous contact after two months in foster home. N o t e t y p i c a l c a l c i f i e d p r i m a r y i n l e f t u p p e r ~obe.
I n a recent discussion (1948) with Dr. Sigrid H o l m of Copenhagen, she inf o r m e d me t h a t for the p a s t eight y e a r s in their series there has not been a ease of tuberculous meningitis in infants who have had successful vaccinations. I n one of Dr. W a l t g r e n ' s r e p o r t s ~'~ he mentions t h a t since the i n a u g u r a t i o n of BCG v a c c i n a t i o n in Gothenburg, over a period of three y e a r s (1942 to 1.945) there was not a d e a t h f r o m tuberculosis in infants u n d e r one y e a r of age. This is strong" evidence t h a t BCG not only p r e v e n t s p r i m a r y infection to a large
ROSENTHAL:
IMMUNOLOGIC
ASPECTS
OF
BCG
417
VACCINATION
]~ig. 18.--:F. R., N e g r o m a l e child, a g e d 2 y e a r s ( v a c c i n a t e d a t 12 d a y s of a g e ) . Note i n f i l t r a t i o n in r i g h t l o w e r lobe. Souree ease, two older children. Continuous contact after s i x w e e k s in. f o s t e r h o m e . H i s s i b l i n g , a c o n t r o l g r o u p child, d i e d f r o m t u b e r c u l o u s m e n i n g i t i s .
l~'ig, 19,--Same
ease as Fig. 18; aged
2 years,
7 months. lobe.
Note
early
calculation
in right
lower"
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extent, but when primary infection does occur, there is less possibility that it will spread hematogenously to other parts of the body. This latter chance is the dreaded and serious aspect of primary tuberculosis. I have recently completed a study of progressive primary tuberculosis in young adults and have shown that there is miliary spread in practically every instance, as shown by post-mortem examination and histologie study. 29 This is also considered to be the course in infants and children. The question always arises as to whether the type of pulmonary lesions in infants and children who react to tuberculin and are reinfected is different as compared to those who do not originally react to tuberculin. From the x-ray films (Figs. 17, 18 and ]9) it is difficult to note any striking difference in the end results. The important feature is that there is more likelihood of healing and calcification in the vaccinated group than there is in the control group. Thus it is evident that BCG vaccination does largely prevent primary infection and the sequelae to primary infection. BCG should not be considered a panacea for prevention of tuberculosis. Massive reinfection will break down immunity to most vaccinations. The most important factor in the prevention of disease is removing the source of infection. This is especially true of tuberculosis. BCG should be administered in tuberculous households only if there is separation before and after vaccination. An infant under a year of age is so easily infected that without isolation BCG is not recommended. It is hard to understand why we should expect more of -BCG, let us say, than of typhoid or smallpox vaecinati0n, where in the former stringent efforts are made to minimize the source of contamination, and in both strictest isolation is practiced. SUI~Il~i ARY
From experimental work in animals in my laboratory, as well as from clinical studies by my associates ~' and myself over a period of fifteen years, I can say unequivocally that BCG is harmless and has definite value in preventing primary infection. It also minimizes the hematogenous sequelae which may follow primary infection, including tuberculous meningitis, miliary tuberculosis, etc. BOG vaccination is an important adjunct to the accepted methods of tuberculosis prevention, such as early diagnosis and isolation of the source case. BCG vaccination should not be expected to do more than other forms of vaccination in which all attempts are made to eliminate the source of contagion. A strictly controlled study is presented of infants born of tuberculous parents. Isolation was practiced after birth in the control group infants and vaccinated infants alike. There were 131 vaccinated infants who were followed for 459 person-years, and 128 control group infants followed for 459 person-years. *Drs. E. Leslie, E. L o e w i n s o h n , a n d M. L e p p m a n n .
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IMMUNOLOGIC ASPE(~TS OF BCG VACCINATION
419
In the vaccinated group there were three patients with roentgenographic evidence of tuberculosis; one was hospitalized and recovered. All showed c a t cification by roentgenogram. In the control group there were six children, four of whom were hospitalized, and three of whom died of generalized tuberculosis. BCG vaccination not only decreased the incidence of primary tuberculosis but also staved off the progression and generalization of the process when infection did occur. The multiple puncture method of vaccination is recommended, since it is simple to apply, is devoid of complications, and has produced universal and lasting (at least five and one-half years) conversion of the tuberculin reaction when applied to newkorn infants. REFERENCES 1. (a) I~rause, A. I~.: Conditions Necessary to Arouse Allergic State in Tuberculosis and Immunity Through Fixation of Bacteria, Tubercle 7: 29-37, 1925. (b) Wfllls, K. S.: Tuberculosis Infection; Early Dissemination of Tubercle Bacilli After Intracutaneous Inoculation of Guinea Figs of First Infection, Am. Rev. Tuberc. Ii: 427-438, 1925. 2. Rosenthal, Sol Roy: The General Tissue and IIumoral Response to an Avirulent Tubercle Bacillus, Urbana~ Ill., 1938, U n i v e r s i t y of Illinois Press. 3. Courmont~ J., and Lesieur, C.: Contribution g l~tude de l'Immunit6 Antitubereuleuse. Rdinoculations N d g a t i v e g Compt. rend. Soc. de biol. 64: 882-883, ]908. 4. l~osenthal, Sol l~oy: Use of BCG V a c c i n a t i o n Among Medical and Nursing Students, Hospitals 17: 75-78, 1943. 5. Daniels, Marc: P r i m a r y Tuberculous I n f e c t i o n in Nurses: 5~[anifestatlons and Prognosis, L a n c e t 2: 165-170, 201-204, 244-246, 1944. 6. (a) Smith, T.: Studies in )/iammalian Tubercle Bacilli. III. Description of a Bovine Bacillus From the iCluman Body: A Culture Test for Distinguishing the Human From the Bovine Type of Bacilli, J. IV[. Research 1.3: 253-300~ 1904-1905. (b) Smith, T.: The Reaction Curve of Tubercle Bacilli From Different Sources in Bouillon Containing Different Amounts of Glycerin, J. IV[. Research 13: 405-408, 1904-1905. 7. Von Behring, E.: Beltrag zur Frage der Rindertuberculose-Immunisirung, Beitr. z. exper. Therap. 10: 1-2], 1905. 8. Calmett% A , and Gu~rin, C.: Sur Quelques Propridtds du Baeille Tuberculenx d'Orlglne Bovine, Cultiv~ sur Bile de Boeuf Glycdrin~e, Compt. rend. Acad. d. .so. 149: 716-718, 1909." 9. Calmette~ A., N~gre, L., and Boquet, A.: Eseais de V a c c i n a t i o n du L a p i n s t dn Cobaye centre ] ' I n f e c t i o n Tuberculeuse, Ann. de l ' I n s t . P a s t e u r 36: 625-631, 1922. 10. Calmette, A., Guhrin, C.: Nouvelles Recherches Expdrimentales sur ]a V a c c i n a t i o n des Bovid6s centre la Tuberculose, Ann. de l ' I n s t . P a s t e u r 3{: 553-560, 1920. 11. Wilbert, J.: Vaccination of lKonkeys A g a i n s t Tuberculosis, Ann. de ] ' I n s t . P a s t e u r 39: 641-651, 1925. 12. Fetroff~ S. A , Branch, A., and Steenken, W., Jr.: Study of Baci]]us-Calmette-Gudrin (BCG): Biological Characteristics, Cultural Dissociation and Animal E x p e r t mentation, Am. Rev. Tuberc. 19: 9-46, 1929. 13. (a) Sasano, i~. T., and Bfedlar, E. M.: Studies of Bacillus-Calmette-Gudrin. Strain of Tubercle Bacillus: Effect " i n v i t r o " of E n v i r o n m e n t on Virulence of BCG, Am. Roy. Tuberc. 23: 215-222, 1931. (b) Reed, G. B., e r r , J. H., and Rice, C. E.: Studies in V a r i a b i l i t y of Tubercle Bacilli: BCG, Canad. J. Research 11: 362-377, 1934. (e) Dreyer~ G., and u R. L.: M u t a t i o n and P a t h o g e n i c i t y E x p e r i m e n t s With BC~, L a n c e t 1: 9-15, 1931. 14. Weill-I~allhj B, and Turpin, R.: Premiers Essais de Vaccination Antituberculeuse de l'Infant par le Bacille Calmette-Gudrln (BCG), Bull. et m@m. See. todd. d. hop. de Paris 49: ]589-1603, 1925. 15. Weill-Hall~, B.: Quoted by Kayne, Gregory G., BCG in Western Europe, Am. Rev. Tuberc. 34: 10, 1936. 16. de Assis, Arlindo: Ensinamentos de Dezessete Anos (1927-1944) de Vaclnac~o BCG no Brasil~ ttospltal, Rio de Janeiro 27: 529-543, 1945. 17. Weill-ttall6, B.: 'La Vaccination Antituberculeuse par l ' I n j e c t i o n Sous-Cutande de BCG: Fresse todd. 36: 721-722, 1928.
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THE JOURNAL OF PEDIATRICS
18. Wallgren, A.:
I n t r a d e r m a l Vaccinations W i t h ]3CG Virus, P r e l i m i n a r y Note, J. A.
M. A. 91: 1876-1881, 1928.
19. (a) Rosenthal, Sol Roy: Studies W i t h BOG: The P r e s e n t Method of BCG Cultivation and Vaccine Production P r a c t i c e d at the P a s t e u r /Institute and the Tiee Laboratories, Ant. Rev. Tubere. 35: 678-684~ 1937. (b) Rosenthal, Sol 1%y: The Multiple P u n c t u r e Method of BCG Vaccination, Am. Rev. Tubere. 39: 128, 1939. (e) Rosenthal, Sol Roy: L a V a c c i n a t i o n au BCG par la Methode des Piqures Superfieielles Multiples, Rev. de la tubere. 5: 815, 1939. 20. Rosenthal, Sol Roy: V a c c i n a t i o n au Moyen de UAntig~ne en Poudre (1)ess~ehd aprgs Congdlation) et de la P l a q u e t t e a Piqures Multiples, Ann. de l~Inst. P a s t e u r 75: 497, 1948. 21. N@gre, L., and Bretey, J.: V a c c i n a t i o n p a r le BCG par Scarifications Cutan6es~ Paris~ 1947, Masson & Cie. 22. F,irkhaug, K.: A Spring-Actuated ]~ultiple P u n c t u r e A p p a r a t u s for BOG Vaccination, Am. J. Clin. P a t h . 17: 751-754, 1947. 23. Rosenthal~ Sol Roy: Conservation du Baeille de Cahnette et Gugrin (BCG) par 1)essiccation aprhs Co~gdlation, Ann. de 1 'Inst. P a s t e u r 75: 209, 1948. 24. (a) Wallgren, A.: Calmette-Vaeeination in Sweden, Swedish N a t i o n a l Association A g a i n s t Tuhereulosis, 1947. (Pamphlet.) (b) l~ertzberg, G.: The A c h i e v e m e n t s of BCG Vaeeination~ Oslo, 29~8, J o h a n G r u n d t T a n u m Forlae. (Book.) (e) I{eimbeek, J.: BCG V a c c i n a t i o n in Nurses, Tubercle 29: 84, 1948. (d) Holm, J o h a n n e s : BCG Vaccination iu Denmark, Pub. H e a l t h Rep. 61: 1298-1316, 1946. 25. Boquet, A.~ and Negre, L.: Vaeeinatiolt P r e v e n t i v e Contre la Tuberculose par le BCG p a r A. Calmette~ C. Gu6rin, Paris, 1927, Masson et C~e. 26. (a) Aronson, J. D., and Palmer, C . E . : Experience W i t h BCG Vaccine in the Control of Tuberculosis Among N o r t h A m e r i c a n Indians, Pub. H e a l t h Rep. 61: 802-820, 1946. (b) Ferguson, R. G.: BCG V a c c i n a t i o n in Hospitals and S a n a t o r i a of Saskatchewan, Canad. J. Pub. H e a l t h 37: 435-451, 1946. 27. Sams, General C.: P e r s o n a l Communication. 28. Rosenthal, Sol Roy, Les]ie~ E. I.~ and Loewinsohn, E.: BCG V a c c i n a t i o n in All Age Groups: J. A. M. A. 136: 73-79, 1948. 29. Rosenthal, Sol Roy: I n Publication.
APRIL, 1950
No. 4
Original Communications IMMUNOLOGIC A S P E C T S O F BCG VACCINATION A
STRICTLY CONTROLLED STUDY I N I N F A N T S FROI~[ TUBERCULOUS I-IousEHOLDS
SOL RoY ROSENTrlAL, M.D., ProD. CHICAGO, ILL.
I W A S invited by the American Pediatric Society to discuss BCG vaccine. This is indeed a sign of a change in our thinking. B e f o r e we can consider vaccination against tuberculosis, I should like first to establish the evidence of i m m u n i t y in tuberculosis. There are m a n y who still doubt t h a t such an i m m u n i t y exists. , Here is some of the evidence which is unequivocal. 1. Koch's P h e n o m e n o n . - - I f a normal guinea pig is inoculated intradermally with virulent tubercle bacilli, no visible reaction can be n o t e d for perhaps f r o m seven to f o u r t e e n days. A t that time a small nodule develops at the site o f inoculation, and the draining lymph nodes become enlarged. The nodule becomes necrotic, breaks down, and ulcerates. At the same time the organisms are dispersed t h r o u g h o u t the b o d y of the animal and the animal finally dies of progressive tuberculosis. Oft the other hand, if one inoculates the same dose i n t r a d e r m a l l y into an infected animal which reacts to tuberculin, one will note in from t w e n t y - f o u r to forty-eight hours an intense redness, swelling, and necrosis at the site of inoculation. This necrotic area will slough out, and if smears are t a k e n of the slough, numerous tubercle bacilli m a y be noted. The local lesion m a y go on to healing and scar formation. The draining lymph nodes will not become grossly enlarged, and culturing of these lymph nodes may be negative for tubercle bacilli; at best they would be positive only between one and three weeks after the second inoculation. (In the primarily infected animals, lymph nodes can be shown to contain tubercle bacilli a few hours after intradermal injection. ~) In my own experiments I demonstrated that there is extension from the site of inoculation in a normal animal as early as five minutes after inoculation. 2 The organisms of reinfection are localized at the site of entrance in infected animals, whereas in the noninfeeted animals tubercle bacilli spread rapidly t h r o u g h o u t the body. F r o m the Institution for Tuberculosis ]~esearch, University of Illinois College of Medicine, Chicago 1Yiunicipal Tuberculosis Sanitorium (Tuberculosis Prevention Clinic) a n d l~esearch F o u n d a t i o n . Presented a t the a n n u a l meeting of The American Pediatric Society at Atlantic City, N. J., iViay 5, 1949. 399
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2. Complete Protection of Animals by Immunization.--It has been demonstrated that one tubercle bacillus wili cause progressive tuberculosis in the guinea pig. If tubercle bacilli are applied to the intact or slightly scarified skin of a guinea pig; progressive tuberculosis may develop. However, if such a guinea pig is first inoculated with avirulent tubercle bacilli, and one waits until the tuberculin test becomes positive, and places virulent tubercle bacilli on the intact or slightly scarified skin, complete protection will be afforded. Likewise, small subcutaneous or intravenous injections of from .001 to .0000001 rag. of virulent tubercle bacilli will produce no demonstrable disease in such animals. 3 Most nonsuccessful experiments have employed relatively massive doses for reinfection, e.g., 0.1 or 1 rag., which would Correspond to many million infective doses.
F i g . 1 . - - L u n g , one a n d o n e - q u a r t e r h o u r s a f t e r i n t r a v e n o u s i n j e c t i o n of BCG. T h e sept a l cells a r e s w o l l e n a n d a r r a n g e t h e m s e l v e s a l o n g t h e i n n e r a s p e c t of t h e a l v e o l a r w a l l s . N o t e t h e fiat d a r k - s t a i n i n g n u c l e i of t h e c a p i l l a r y e n d o t h e l i u m in c o n t r a d i s t i n c t i o n . ( H e m a t o x y lin 'and e o s i n X 5 0 0 . )
3. Hyperergy and Resistance to Tuberculosis in Human Beings.---If a natural primary infection in a young adult is completely healed (as depicted by a positive tuberculin test and a negative chest film), it has been shown repeatedly that this person resists the tuberculous reinfection to a much higher degree than one who has not had the primary infection. This has been shown in one of our own studies of various institutions ill the United States ~ and has been enlarged upon by Mare Daniels in a survey of some twenty reports from many parts of the world2 He found that in more than 8,000 student nurses who were initially tuberculin positive, ]73 or 2.1 per cent of them developed tu-
ROSENTHAL:
I M M U N O L O G I C A S P E C T S OF BCG V A C C I N A T I O N
401
berculosis in contrast to 5.8 per cent of 4,832 nurses wnose tuberculin reactions were initially negative. His own survey in England of more than 2,500 nurses was 2 per cent versus 6 per cent. In oontrast, positive tuberculin reactions in infants and children more frequently indicate active infection, which may or may not progress. The interpretation of a positive tuberculin reaction differs sharply with age.
4. The Retieulo-Endothelial System and Immunity in Tuberculosis.--The nature of immunity to tuberculosis is not entirely understood. It is generally agreed that it is not humoral, but rather connected with the cellular responses, particularly those of the rectieulo-endothelial system. I have shown 2 that the introduction into the guinea pig of avirulent tubercle bacilli, whether by the parenteral or, to a lesser extent, by the enteral route, is followed by a swelling and proliferation of the reticulo-endothelial system, especially in the lung (Figs. 1, 2, 3, and 4). This phenomenon is manifest shortly after inoculation, pro-
F i g . 2 . - - L u n g , f o r t y - e i g h t h o u r s a f t e r i n t r a v e n o u s i n j e c t i o n of ]BCG. A t u b e r c l e c o m p o s e d of a c e n t r a l g r a n u l o e y t e a n d i n t r a a e l l u l a r t u b e r c l e bacilli a n d a p r e p o n d e r a n c e of e p i t h e l i o i d cells in t h e p e r i p h e r y . ( H e m ' a t o x y l i n a n d e o s i n XS00.)
gresses rapidly for a week or two, and then gradually recedes. The injection of large doses of tuberculin (10 rag. intracutaneously) produces a severe local reaction. The reticulo-endothelial system, especially of the lung, will swell, and a marked hyperemia accompanies the swelling (Fig. 5). The blood monocyte response trails the tissue reaction. The m/1 (monoeyte/lymphocyte) ratio is increased early and during the production of the local Koch's phenomenon (Fig. 6).
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Fig'. 3 . - - L i v e r , s i x t e e n h o u r s a f t e r i n t r a v e n o u s i n j e c t i o n of ]~CG. N o t e p r o l i f e r a t i o n of t h e K u p f f e r ceils a n d t h e g r o u p i n g of s a m e i n t o s m a l l n o d u l e s . S o m e o f t h e cells c o n t a i n e d tubercle bacilli. (Hematoxylin and eosin XS00.)
F i g . 4 . - - K i d n e y , o n e a n d o n e - q u a r t e r h o u r s a f t e r i n t r a v e n o u s i n j e c t i o n of BCG, P e r t pelvic fat showing marked perivascular histiocytic mobilization and proliferation. (Hematoxylin and eosin X500.)
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HISTORY OF DEVELOPMENT OF BCG Theobald Smith s was one of the first to show t h a t the bovine tubercle bacillus differs f r o m the h u m a n t y p e bacillus. H u m a n tubercle bacilli are less v i r u l e n t for cattle t h a n for humans, and, conversely, bovine tubercle bacilli are less v i r u l e n t for h u m a n s t h a n for cattle. W o r k i n g on this principle, Von B e h r i n g 7 used h u m a n tubercle bacilli, which he a t t e n u a t e d b y culturing and aging, to vaccinate calves. The results were most e n c o u r a g i n g in t h a t tuberculosis was prevented, b u t it was soon discovered t h a t v a c c i n a t e d cows would excrete h u m a n tubercle bacilli in t h e i r milk, a n d the vaccinations were a b r u p t l y stopped.
F i g . 5 . - - L u n g , t h r e e m o n t h s a f t e r i n t r a v e n o u s i n j e c t i o n of t~CG. M o d e r a t e h y p e r e m i ' a a n d s e p t a l cell s w e l l i n g f o l l o w i n g a K o c h ' s p h e n o m e n o n d u e to old t u b e r c u l i n . (Heraatoxylin and eosin X125.)
A t the same time Calmette and Gu6rin, who were t h e n at Lille, b e g a n w o r k i n g w i t h the idea t h a t bovine tubercle bacilli m i g h t be used to immunize humans. Their w o r k b e g a n in 1908, s using an organism which was a l r e a d y iu the l a b o r a t o r y and h a d originally been isolated f r o m the milk of a tuberculous cow. T h e y b e g a n to a t t e n u a t e this organism b y growing it on ox bile potato media, which t h e y showed r e d u c e d the virulence of tubercle bacilli. A f t e r five y e a r s of such cultivation it was n o t e d t h a t this organism, which h a d originally been capable of p r o d u c i n g progressive tuberculosis in laborat o r y animals, n o w failed to do so. W h e n it was n o t e d a f t e r m a n y more y e a r s t h a t this p r o p e r t y of avirulence was constant, i m m u n i z a t i o n of animals was
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begun, first in small l a b o r a t o r y animals, 9 then in cattle, 1~ and finally in monkeys and apes. 11 Some of the early classical experiments were done as follows : Gu6rin was a veterinarian, and since the problem of bovine tuberculosis was always acute in France, Calmette and Gu6rin concentrated their earlier work on cattle. They placed five adult tuberculous cows in one row of stalls, and in the next row behind them ten calves. Six of the calves received 20 mg. of the a t t e n u a t e d organism intravenously, and the other four served as controls. The vaccinated and control calves were placed alternately. The tuberculous cows were moved every week so t h a t contamination would be uniform. A f t e r eighteen months of the experiment, none of the six vaccinated
3 Mqnths (No. 17)
I t
,4
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! ,4W :.-d-.. 8~ I l l
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/
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:
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o :'; ~ "
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9
"...o.
,
V ==.
o'" ]~
O~,y~,
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"oo.o.. ~
Tuberculin Te~t Positive
,. M o r l o c ~ e ~ ~
4
l~ig. 6 . - - T h e b l o o d r e s p o n s e f o l l o w i n g t h e i n t r a v e n o u s m o n o c y t e s in t h e b l o o d i n c r e a s e d f o l l o w i n g t h e m o b i l i z a t i o n m/1 r a t i o ( m o n o c y t e / l y m p h o c y t e ) increased during Koch's tuberculin.
6
6
w e e I.<0
I~
injection of BCG. The total of t h e t i s s u e h i s t i o c y t e s . The phenomenon p r o d u c e d b y old
cows r e a c t e d to tuberculin, whereas three of the f o u r controls were infected.
After thirty-two months, autopsy of all the animals revealed that three of the four controls presented extensive tuberculous lesions in the lungs and lymph nodes, only one of the vaccinated showed a small lesion in the lung and lymph nodes, while the others showed no lesions whatsoever. A similar experiment was done on monkeys. Unfortunately, the experiments were only partly successful since so many of the animals were tuberculous when they arrived in Europe. The project was moved to French Guinea, the natural habitat of the monkeys, and the same type of experiment was done. Tuberculous animals, vaccinated animals, and nonvaccinated animals were
ROSENTHAL:
I~IUNOLOGIC
A S P E C T S OF BC(~ V A C C I N A T I O N
405
housed in the same cage. Practically all of the control monkeys and chimpanzees died os tuberculosis, whereas none of the vaccinated animals showed signs of the disease. 11 After numerous similar experiments it was definitely established that the attenuated organism did not produce progressive disease in animals, and that it would protect against natural infection by virulent tubercle bacilli. Such was the beginning of the bacillus Calmette and Gu6rin (BCG). THE BACILLUS
Now that I have discussed some of the immunological properties of the organism, I wish to discuss some of the properties of the bacillus itself. It behaves much the same as a bovine tubercle bacillus on culture. It takes about a week or ten days to grow on egg media, and produces coiled colonies which are butter yellow in color. If special methods are used, however, it is very simple to dissociate this organism into a smooth (Fig. 7) and a rough form (Fig. 8). The accompanying figures will demonstrate the difference in morphology.
Fig.
7.
Fig.
8,
Fig. 7 . - - S m o o t h " S " B C G colonies. N o t e e d g e s s l o p i n g into m e d i u m ( D E 7.2). A g e of c u l t u r e , 20 d a y s . Source, o r g a n s of i n f e c t e d ' a n i m a l s . F i g . 8 . - - R o u g h "1~" B C G colonies on L b w e n s t e i n ' s m a l a c h i t e g r e e n e g g m e d i u m ( p H 6.1). T h i s is t h e t w e l f t h g e n e r a t i o n f r o m a n o r i g i n a l blood c u l t u r e . A g e of colony, 5 d a y s . N o t e t h e s i n g l e l a r g e g r a n u l a r colonies, t h e coiled a n d t h e c r a t e r l i k e o n e s c o m p o s e d of s i m i l a r g r a n u l e s , T h e d e m a r c a t i o n of c o l o n y a n d m e d i u m is d i s t i n c t .
Neither type of colony will produce progressive tuberculosis if inoculated into guinea pigs. We have inoculated thousands of animals, and we have never seen progressive tuberculosis following BCG inoculation. Some of the early work done by Petroff 1~ and others, 13 in which they claimed that the dissociate strains were virulent, has not been repeatable since then. The morphology of the organism is of special interest. It is an acid-fast curved rod much the same as the bovine tubercle bacillus (Fig. 9). Using special methods, including the electron microscope, shadow-casting, and hyper-
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tonic solutions, certain characteristics of the o r g a n i s m have been disclosed which are of p a r t i c u l a r interest. I found (with Dr. C. I. Reed and B. P. Reed) that the bacillus has a definite capsule (Fig. 10), and granules can be f o u n d (Fig. 11) which are, most likely, the origin of new bacilli. BCG is an a v i r u l e n t bovine tubercle bacillus which retains the cultural and morphologic characteristics of bovine m y c o b a c t e r i u m .
Fig.
9,--Bacillus Calmette-Gudrin showing smooth surface and pol'ar bodies. Electronmicro~ scopic photo following siladow casting with chromium. (X45,500.)
F i g . l O . - - B a e i l l u s C a t m e t t e - ~ u d r i n a f t e r s h r i n k i n g in h y p e r t o n i c s o l u t i o n a n d s h a d o w Note definite capsule und shadow cast by irregular surface. (X45,500.)
casting.
VACCINATION
Vaccination in Humans.--After n u m e r o u s studies in animals, and a f t e r showing the avirulence of the organism, Calmette and Gu6rin were still rel u c t a n t to use it in humans. I t was Well-Hall614 who was allowed b y Professor Calmette to use the vaccine first in h u m a n s ; this was e a r l y in 1921, a f t e r there h a d been 230 consecutive t r a n s f e r s f r o m the original s t r a i n over a period of t h i r t e e n years. Methods of Vaccination.--The question arose at once as to the best method of v a c c i n a t i n g humans. Since B e s r e d k a ' s influence was s t r o n g at this time, it was decided to use the oral route. I t was soon learned, however, t h a t the
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I M M U N O L O G I C ASPECTS OF BCG V A C C I N A T I O N
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rate of conversion of the tuberculin reaction following this method of vaccination was very low; if ]0 rag. of BCG were given orally on the third, fifth, and seventh day of life, a positive yon Pirquet's test occurred in only ]5 per cent of the infants after three months. More recently in Brazil, Arlindo de Assis has been using massive doses of vaccine by mouth. ~G He has given as much as 90 rag. in the first 10 days of life, and has reported 83 per cent positive reactors after the fourth month (though he used 10 rag. of 0. T. as a test dose).
Fig.
ll.--Bacillus
Calmette-Gudrin.
Same treatment (•
as
in F i g .
10.
N o t e definite g r a n u l e s .
Weill-Hall6,17 using the subcutaneous method, obtained 100 per cent conversion after nine weeks by the administration of from .02 to .04 rag. of vaccine. Unfortunately, there were many complications, including cold abscesses which persisted for long periods of time. Wallgren TM later suggested and used the intradermal route, and this method produced a high degree of conversion; superficial ulcers occurred in a high percentage of the cases, however, and if the vaccine was not administered properly, small abscesses occasionally resuited. One must be extremely careful with this method in order to avoid complications. Wa]lgren, using 0.1 rag., noted 100 per cent positive reactors after seventeen weeks. In an effort to avoid complications, I described the scarification method of multiple punctures in 1937. 29 The principle of this method was the introduction of minute doses of vaccine at multiple sites into the superficial layers
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of the skin transcutaneously, r a t h e r than a single large dose intradermally, which so often leads to necrosis and slough. In the original procedure a drop of vaccine was placed on the skin, and t h i r t y punctures were made with a sewing-type needle through the vaccine into the skin over an area 3 • 11/2 cm. square.
2'ig. 1 2 . - - F a p u l e s
t e n d a y s following" v a c c i n a t i o n in a young" a d u l t .
Note
discreteness.
R e c e n t l y we have developed a stainless steel disk 1 inch in diameter in which there are thirty-six prong's and which can be controlled by a small magnet. 2~ The entire vaccination is accomplished with one pressure. A week or so a f t e r this m e t h o d is used, small 2 to 3 ram. papules develop (Fig. 12), remain s t a t i o n a r y for f r o m two to three weeks, then regress and heal without gross evidence of scarring. I n infants the lesions are minute, about 1 ram. in diameter, and so/netimes difficult to see; in fact, the m o t h e r r a r e l y knows on which arm the child was vaccinated. The draining l y m p h nodes m a y become slightly enlarged, and m a y be p a l p a t e d if a special effort is made. T h e y never enlarge to become grossly visible, and never b r e a k down. The original method is modified in F r a n c e 21 b y making numerous scratches t h r o u g h a drop of vaccine, and in New York b y the spring-actuated multiple p u n c t u r e a p p a r a t u s of Birkhaug. 22
Technique of Using Multiple Puncture Disk.--Cleanse the skin over the deltoid region of the left arm with acetone and allow it to d r y thoroughly. Place a drop of vaccine on the arm with a capillary tube or from an ampule (Fig. 13). Grasp the disk with the alcohol-cleansed metal plate of the magnet, allowing the wide margin of the d~sk to e x t e n d b e y o n d the m a g n e t and away f r o m the operator. It should be held at a 30 degree angle. Tap the drop of
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I1V[1V~UNOLOGIC
A S P E C T S OF BCG V A C C I N A T I O N
409
vaccine with the wide margin of the disk, and pull it along so that an area 2.5 cm. in diameter is covered with vaccine. Dip the points of the disk into the vaccine, rotating the disk slightly so that the points become moistened.
Fig. 13.--The "set up" for vaccinating.
Note saw, capillary tube with vaccine, disk, and magnet.
l~ig. 14.--~r of u s i n g m u l t i p l e p u n c t u r e d i s k . N o t e p o s i t i o n - - h o l d e r in p a l m J u s t beneath the index finger, and the other hand grasping the arm underneath, and tensing skin. T h e d i s k is h e l d in p l a c e b y m a g n e t i c p l a t e .
Place the disk in the center of of the vaeeine and move the metal plate of the magnet so that it is now centered on the disk; This will avoid bending the metal. With the butt of the magnet in the palm, just beneath the index finger, and with the other hand grasping the arm underneath and tensing the skin appreciably (Fig. 14), apply pressure so that the points of the disk are well buried in the skin. In adults considerable pressure is used, whereas in newly born and older infants only enough pressure is applied so that the penetration of the points is readily felt through the palm of the operator's hand. With the pressure still exerted as above, rock the magnet and disk forward and backward and from side to side several times. Be sure to maintain the original pressure. At this point release the grasp on arm and slide the
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magnet away, off the disk, and toward the operator, maintaining the pressure, though slightly less than originally.' Following a successful procedure the disk remains on the arm, lying flat, with the points still buried in the skin. If the points are on top of the skin, then the procedure must be repeated. With the thumb and index finger, grasp the disk at the edge opposite the wide border and gently lift it away from the skin. There should be a feeling of the points disengaging themselves from the skin. Tap the vaccine again with the wide margin of the disk so that each perforation of the skin is covered with vaccine.
F i g . 1 5 A . - - S i t e of i n j e c t i o n f o l l o w i n g m u l t i p l e p u n c t u r e t e c h n i q u e . Note minute lesion just beneath epithelium and 'absence of dilated polymorphonuclear-fllled lymphatics. (Guinea p i g s k i n , five h o u r s a f t e r i n j e c t i o n . )
Do not use much pressure, since it will press the vaccine out of the perforations. Allow the vaccine to dry on the arm without dressing. Do not wash the arm for twenty-four hours. No other precautions are necessary. The disks are inexpensive and may easily be sterilized by boiling. The microscopic lesions produced by the multiple puncture method are of interest in that the vaccine enters into the most superficial layers of the skin, usually just beneath the epidermis. Since only small doses are delivered at any one point, there is never any abscess formation. Because the vascular
~R0~ENTttAL:
IMMUNOLOGIC ASPECTS OF BCG VACCINATION
411
supply at this level in the skin is relatively great and the ceils of the retieuloendothelial system are most numerous, many tubercle and giant cells form (Figs. 15, A and B).
Fig. 1 5 B . - - N u m e r o u s g i a n t cells and histiocytie proliferation, ten days a f t e r injection.
In contrast, it is ahnost impossible to inject material by intradermal injection as superficially as by the transcutaneous method, and the vaccine is introduced by that method into the portion of the skin which is poor in blood supply and reticulo-endothelial cells. Because of the relatively large dose at one point a small area of necrosis forms which is surrounded by a cuff of pus cells which partially occludes the lymphatics and further favors central necrosis (Figs. 16, A and B). Since this necrotic material must either be extruded or absorbed, ulcers form unless absorption takes place. The Rapidity of Development of Tuberculin Reactions.~It is possible to produce a positive tuberculin test in guinea pigs in from one to two weeks following a single puncture through a drop of vaccineY a In newborn infants,
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8. F i g . 1 6 . - - A , Site of i n j e c t i o n f o l l o w i n g c a r e f u l s u p e r f i c i a l i n t r a d e r m a l inoculatiorf ( g u i n e a p i g ) . N o t e t h a t c e n t e r of l e s i o n is d e e p in t h e r e t i c u l u m l a y e r 'and n e c r o t i c ( 1 ) ; a n d a b r o a d zone of p o l y m o r p h o n u e l e a r i n f i l t r a t i o n s~w~ouncls site ( 2 ) . (l~our h o u r s a f t e r i n j e c t i o n h e m a toxylin a n d eosln xSO.) 13, I/]dge of lesion showing" in (A). Note that the Polymorphonuclear cells are in dilated lymphatics. (l-Iematoxylin a n d eosin XI$O.)
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children, and adults we have tried f r o m t h i r t y to n i n e t y punctures, and v a r y ing concentrations of vaccine, but at the p r e s e n t time we employ the multiple p u n c t u r e disk and vaccine which contains 15 mg./ml, moist weight of organisms. Positive tuberculin reactions a p p e a r as early as eleven to thirteen days following vaccination in infants, a n d they are almost u n i v e r s a l l y positive at one month. W e m a y get as high as 90 per cent conversion in older child r e n and adults in two w e e k s ' time. The complications following this method are p r a c t i c a l l y nil.
Immunity Following ]Taccination.--I will not go into the literature deeply in discussing the i m m u n i t y c o n f e r r e d b y BGG vaccination. All of y o u are f a m i l i a r with the excellent w o r k of the Scandinavians, 24 the French, 25 the Americans, ~6 and a host of others all over the world. Up to the p r e s e n t time there have been almost 40 million vaccinations p e r f o r m e d , 30 million in J a p a n alone. 27 The n u m b e r done in Russia is not known, but it would p r o b a b l y g r e a t l y enlarge these figures. There is u n a n i m i t y in p r a c t i c a l l y every r e p o r t t h a t a definite r e d u c t i o n in the m o r b i d i t y and m o r t a l i t y of tuberculosis follows B C G vaccination. M a n y of the reports have been criticized because of the lack of controls. W e have t r i e d to set up e x p e r i m e n t s in Chicago which would meet all the criticisms concerning contro]s which h a v e been m a d e in the past. as I n this s t u d y our control group subjects have been selected in as u n b i a s e d a m a n n e r as we t h i n k possible. These infants were exclusively f r o m tuberculous tim> ilies. T h e y were r e f e r r e d to us b y the Chicago municipal tuberculosis clinics and b y p r i v a t e physicians a n d clinics. The diagnostic d a t a and the results of the s p u t u m e x a m i n a t i o n of the tuberculous contact were submitted, and all x-rays were reviewed b y our clinic before the diagnosis was accepted. W i t h the b i r t h of the child, i m m e d i a t e s e p a r a t i o n was a r r a n g e d if the m o t h e r was t u b e r c u l o u s ; if other m e m b e r s of the f a m i l y were tuberculous, the child was allowed to s t a y in the hospital as long as the m o t h e r was there. ]n e v e r y ease the child was t h e n placed in one of our own foster homes. Visits to the foster home were m a d e b y our p e d i a t r i c i a n e v e r y week. The vaccin,~tion or placebo was done in the foster home. The d u r a t i o n of isolation r a n g e d ft'om six weeks to four and one-half months, d e p e n d i n g u p o n the t y p e of ease. W h e r e the m o t h e r had f a r a d v a n c e d disease and a positive sputum, for example, the child was not v a c c i n a t e d until three months a f t e r birth, p r o v i d i n g there was a n e g a t i v e x - r a y and a negative tuberculirt test. W h e n it was the f a t h e r who was tuberculous, the child was vaccinated three to seven days after birth. I n f a n t s were usually r e t u r n e d to their homes six weeks a f t e r vaccination, p r o v i d e d the M a n t o u x test had become positive in v a c c i n a t e d individuals, or' if it was n e g a t i v e in children who were controls and there was no open source ease. E i g h t y per cent were r e t u r n e d in this manner. The division of eases was done b y a m a s t e r chart. The doctor who did the v a c c i n a t i n g n e v e r m a d e the choice as to w h e t h e r the child was to be a contr'o] or vaccinated. The eases were strictly a l t e r n a t e d in categories d e t e r m i n e d b y
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the types of source cases. Children w h o were to return to a tuberculous env i r o n m e n t were v a c c i n a t e d w i t h twice the dose used for the n o n c o n t a c t infants (i.e., t w o series of thirty punctures with the straight needle, or, more recently, a series of two disk punctures). Progress of the infants w a s f o l l o w e d by means of clinic visits at approximately six-month intervals; t h e y were theu tuberculin tested, x-rayed, and the a m o u n t of contact w h i c h they had had during the preceding period w a s ascertained. B e t w e e n the clinic visits the infants were seen at intervals by a nurse in the home. It m a y be seen in the table that there were 139 infants in the vaccinated group and _128 in the control group. There were practically the same number of v a c c i n a t e d and control group infants in each c a t e g o r y (Table I), according to the type of tuberculosis in the source case. The a m o u n t of actual contact was similar, tho.ugh the vaccinated group had the more contact (186 person years contact with the mother in the vaccinated group as against _160 in the controls) (Table II). TABLE I .
~OURCE OF ~ONTACT AND TYPE OF TUBEI~CULOSIS AT ]~IRTH OF ~HII,D
MINIMAL
ATED
CO~TROL
11
]1
43
10
9
18
VACCIN-
CONTACT SOURCE
Mother (Alone or with others) Father (Alone or with others)
MODEgATELY ADVANCED VACCIN- GONATED TROL
POSITIVE SPUTU~
FAg ADVANCED VAGCINATED
CONTgOL
VACCINATED
CONTROL
41
33
36
27
24
17
17
14
14
14
F o l l o w i n g v a c c i n a t i o n there was -100 per cent tuberculin conversion, and this remained constant for almost five and one-half years (Table III). In the control group the number of individuals at each age was practically the same as for the vaccinated. F o r example, at birth there were 131 and 128, respectively; at ]9 to 24 months, there were 102 and 100; and at 61 to 66 months, there were 40 and 40. The degree of exposure is evident w h e n it is noted that at 61 to 66 months, 57.6 per cent of the n o n v a c c i n a t e d control group infants reacted to tuberculin ( V o l l m e r patch test) (Table I V ) . The a m o u n t of contact must have been great since in our so-called n o n c o n t a c t group only 32.9 per cent of the infants had a positive reaction at that age, and this n o n c o n t a c t group of infants lived in the most h e a v i l y infected district of Chicago? s TABLE ]-I. AMOUNT OF CONTACT W I T t I :PULMONARY TUBEI~CULOSIS VACCINATED
Continual~ w i t h mother Average Continual~ w i t h father Average Continual~ w i t h others Average Intermittent, w i t h any Average
186.5 2.8 72.2 2.8 11.9 1.3 37.3 2.2
person-years years person-years years person-years years person-years years
I
CONTROLS
160.1 2.6 73.1 3.3 25.1 2.5
person-years years person-years years person-years years 56.2 person-years 2.4 years
ROSENTEAL: TABLE
III.
I1K1Vs
TUBERCULIN
I~EACTIONS
]FOLLOWING A SINGLE GROUP OF INFANTS
AGE (]Y[O.)
NO. REGISTERED
NO. TESTED
0-3 4-7 8-12 13-18 19-24 25-30 31-36 37 42 43-48 49-54 55-60 61-66
131 123 I18 111 102 91 78 72 63 ~4 48 40
124 116 107 95 87 77 69 59 52 43 36 27
TABLE
IV.
AGE
(MO.)
0-3 4-7 8~12 13-18 19-24 25-30 31-36 37-42 43-48 49-54 55-60 61-66
TUBERCULIN
415
ASPECTS OF BCG VACCINATION
ID~EACTIONS IN THE NO.
NO. POSITIVE
124 116 107 95 87 77 68 58 51 42 35 26
CONTACT
CONTROL
NO.
REGISTERED
TESTED
128 124 115 109 100 93 86 79 73 66 52 40
128 120 110 100 91 83 75 70 64 53 43 33
IN THE
VACCINATION
CONTACT
% POSITIVE
100 100 100 ]00 100 100 98.6 98.3 98.1 97.7 97.2 96.3
GROUP
OF INFANTS
NO.
v/o
POSITIVE
POSITIVE
0 9 20 41 45 46 42 41 36 31 24 19
0 7.5 18.2 41 49.5 55.4 56 58.6 56.3 58,5 55.8 57.0
There were twice the n u m b e r of cases of tuberculosis in children in the control group as a m o n g the v a c c i n a t e d (six cases against three) (Table V). W h a t is more significant is the f a c t t h a t only one child f r o m the vaeci~lated group needed hospitalization, while four f r o m the control group required it! Indeed, in the v a c c i n a t e d group, two of the children were diagnosed as tuber' eulous only because of calcified lesions in the lung; the t h i r d showed a small area of infiltration in the left hilus which calcified in a short period of time. TABLE V. INCIDENCE OF TUBERCULOSIS IN THE CONTACT GROUP OF CHILDREN
Total number of children Person-years observed Cases of tuberculosis Total number Rate "x Children hospitalized Total number :Rate ~ D e a t h s from t u b e r c u l o s i s Total number R a t e "X" Deaths from other causes Total number :Rate ~ *Rate per t h o u s a n d p e r s o n - y e a r s observation,
VACCINATED 131 459 3 6.5
i
CONTROLS 128 459 6 13
1 2.2
4 8.7
0 0
3 6.5
2 4.3
2 4,3
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On the other hand, t h r e e of the six children in the control group died. All were autopsied and were f o u n d to h a v e a t y p i c a l p r i m a r y lesion in the lung w i t h s p r e a d to tile hilus l y m p h nodes; in one ease there was a m i l i a r y tuberculosis and tuberculous meningitis. These f e a t u r e s are most striking, sinee it a p p e a r s t h a t BCG Vaccination m a y p r e v e n t v i r u l e n t tubercle bacilli f r o m establishing themselves in the host, or if the infecting dose is large and they do get a foothold, their s p r e a d f r o m the site of entrance is inhibited. I t is m u c h the same as I have described in e x p e r i m e n t a l animals.
Fig. 17.~D. F., a w h i t e f e m a l e c h i l d v a c c i n a t e d a t 5 w e e k s o f a g e . X-ray taken at 5 years of age. Mother and fa.ther both tuberculous. Continuous contact after two months in foster home. N o t e t y p i c a l c a l c i f i e d p r i m a r y i n l e f t u p p e r ~obe.
I n a recent discussion (1948) with Dr. Sigrid H o l m of Copenhagen, she inf o r m e d me t h a t for the p a s t eight y e a r s in their series there has not been a ease of tuberculous meningitis in infants who have had successful vaccinations. I n one of Dr. W a l t g r e n ' s r e p o r t s ~'~ he mentions t h a t since the i n a u g u r a t i o n of BCG v a c c i n a t i o n in Gothenburg, over a period of three y e a r s (1942 to 1.945) there was not a d e a t h f r o m tuberculosis in infants u n d e r one y e a r of age. This is strong" evidence t h a t BCG not only p r e v e n t s p r i m a r y infection to a large
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IMMUNOLOGIC
ASPECTS
OF
BCG
417
VACCINATION
]~ig. 18.--:F. R., N e g r o m a l e child, a g e d 2 y e a r s ( v a c c i n a t e d a t 12 d a y s of a g e ) . Note i n f i l t r a t i o n in r i g h t l o w e r lobe. Souree ease, two older children. Continuous contact after s i x w e e k s in. f o s t e r h o m e . H i s s i b l i n g , a c o n t r o l g r o u p child, d i e d f r o m t u b e r c u l o u s m e n i n g i t i s .
l~'ig, 19,--Same
ease as Fig. 18; aged
2 years,
7 months. lobe.
Note
early
calculation
in right
lower"
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extent, but when primary infection does occur, there is less possibility that it will spread hematogenously to other parts of the body. This latter chance is the dreaded and serious aspect of primary tuberculosis. I have recently completed a study of progressive primary tuberculosis in young adults and have shown that there is miliary spread in practically every instance, as shown by post-mortem examination and histologie study. 29 This is also considered to be the course in infants and children. The question always arises as to whether the type of pulmonary lesions in infants and children who react to tuberculin and are reinfected is different as compared to those who do not originally react to tuberculin. From the x-ray films (Figs. 17, 18 and ]9) it is difficult to note any striking difference in the end results. The important feature is that there is more likelihood of healing and calcification in the vaccinated group than there is in the control group. Thus it is evident that BCG vaccination does largely prevent primary infection and the sequelae to primary infection. BCG should not be considered a panacea for prevention of tuberculosis. Massive reinfection will break down immunity to most vaccinations. The most important factor in the prevention of disease is removing the source of infection. This is especially true of tuberculosis. BCG should be administered in tuberculous households only if there is separation before and after vaccination. An infant under a year of age is so easily infected that without isolation BCG is not recommended. It is hard to understand why we should expect more of -BCG, let us say, than of typhoid or smallpox vaecinati0n, where in the former stringent efforts are made to minimize the source of contamination, and in both strictest isolation is practiced. SUI~Il~i ARY
From experimental work in animals in my laboratory, as well as from clinical studies by my associates ~' and myself over a period of fifteen years, I can say unequivocally that BCG is harmless and has definite value in preventing primary infection. It also minimizes the hematogenous sequelae which may follow primary infection, including tuberculous meningitis, miliary tuberculosis, etc. BOG vaccination is an important adjunct to the accepted methods of tuberculosis prevention, such as early diagnosis and isolation of the source case. BCG vaccination should not be expected to do more than other forms of vaccination in which all attempts are made to eliminate the source of contagion. A strictly controlled study is presented of infants born of tuberculous parents. Isolation was practiced after birth in the control group infants and vaccinated infants alike. There were 131 vaccinated infants who were followed for 459 person-years, and 128 control group infants followed for 459 person-years. *Drs. E. Leslie, E. L o e w i n s o h n , a n d M. L e p p m a n n .
ROSENTHAL:
IMMUNOLOGIC ASPE(~TS OF BCG VACCINATION
419
In the vaccinated group there were three patients with roentgenographic evidence of tuberculosis; one was hospitalized and recovered. All showed c a t cification by roentgenogram. In the control group there were six children, four of whom were hospitalized, and three of whom died of generalized tuberculosis. BCG vaccination not only decreased the incidence of primary tuberculosis but also staved off the progression and generalization of the process when infection did occur. The multiple puncture method of vaccination is recommended, since it is simple to apply, is devoid of complications, and has produced universal and lasting (at least five and one-half years) conversion of the tuberculin reaction when applied to newkorn infants. REFERENCES 1. (a) I~rause, A. I~.: Conditions Necessary to Arouse Allergic State in Tuberculosis and Immunity Through Fixation of Bacteria, Tubercle 7: 29-37, 1925. (b) Wfllls, K. S.: Tuberculosis Infection; Early Dissemination of Tubercle Bacilli After Intracutaneous Inoculation of Guinea Figs of First Infection, Am. Rev. Tuberc. Ii: 427-438, 1925. 2. Rosenthal, Sol Roy: The General Tissue and IIumoral Response to an Avirulent Tubercle Bacillus, Urbana~ Ill., 1938, U n i v e r s i t y of Illinois Press. 3. Courmont~ J., and Lesieur, C.: Contribution g l~tude de l'Immunit6 Antitubereuleuse. Rdinoculations N d g a t i v e g Compt. rend. Soc. de biol. 64: 882-883, ]908. 4. l~osenthal, Sol l~oy: Use of BCG V a c c i n a t i o n Among Medical and Nursing Students, Hospitals 17: 75-78, 1943. 5. Daniels, Marc: P r i m a r y Tuberculous I n f e c t i o n in Nurses: 5~[anifestatlons and Prognosis, L a n c e t 2: 165-170, 201-204, 244-246, 1944. 6. (a) Smith, T.: Studies in )/iammalian Tubercle Bacilli. III. Description of a Bovine Bacillus From the iCluman Body: A Culture Test for Distinguishing the Human From the Bovine Type of Bacilli, J. IV[. Research 1.3: 253-300~ 1904-1905. (b) Smith, T.: The Reaction Curve of Tubercle Bacilli From Different Sources in Bouillon Containing Different Amounts of Glycerin, J. IV[. Research 13: 405-408, 1904-1905. 7. Von Behring, E.: Beltrag zur Frage der Rindertuberculose-Immunisirung, Beitr. z. exper. Therap. 10: 1-2], 1905. 8. Calmett% A , and Gu~rin, C.: Sur Quelques Propridtds du Baeille Tuberculenx d'Orlglne Bovine, Cultiv~ sur Bile de Boeuf Glycdrin~e, Compt. rend. Acad. d. .so. 149: 716-718, 1909." 9. Calmette~ A., N~gre, L., and Boquet, A.: Eseais de V a c c i n a t i o n du L a p i n s t dn Cobaye centre ] ' I n f e c t i o n Tuberculeuse, Ann. de l ' I n s t . P a s t e u r 36: 625-631, 1922. 10. Calmette, A., Guhrin, C.: Nouvelles Recherches Expdrimentales sur ]a V a c c i n a t i o n des Bovid6s centre la Tuberculose, Ann. de l ' I n s t . P a s t e u r 3{: 553-560, 1920. 11. Wilbert, J.: Vaccination of lKonkeys A g a i n s t Tuberculosis, Ann. de ] ' I n s t . P a s t e u r 39: 641-651, 1925. 12. Fetroff~ S. A , Branch, A., and Steenken, W., Jr.: Study of Baci]]us-Calmette-Gudrin (BCG): Biological Characteristics, Cultural Dissociation and Animal E x p e r t mentation, Am. Rev. Tuberc. 19: 9-46, 1929. 13. (a) Sasano, i~. T., and Bfedlar, E. M.: Studies of Bacillus-Calmette-Gudrin. Strain of Tubercle Bacillus: Effect " i n v i t r o " of E n v i r o n m e n t on Virulence of BCG, Am. Roy. Tuberc. 23: 215-222, 1931. (b) Reed, G. B., e r r , J. H., and Rice, C. E.: Studies in V a r i a b i l i t y of Tubercle Bacilli: BCG, Canad. J. Research 11: 362-377, 1934. (e) Dreyer~ G., and u R. L.: M u t a t i o n and P a t h o g e n i c i t y E x p e r i m e n t s With BC~, L a n c e t 1: 9-15, 1931. 14. Weill-I~allhj B, and Turpin, R.: Premiers Essais de Vaccination Antituberculeuse de l'Infant par le Bacille Calmette-Gudrln (BCG), Bull. et m@m. See. todd. d. hop. de Paris 49: ]589-1603, 1925. 15. Weill-Hall~, B.: Quoted by Kayne, Gregory G., BCG in Western Europe, Am. Rev. Tuberc. 34: 10, 1936. 16. de Assis, Arlindo: Ensinamentos de Dezessete Anos (1927-1944) de Vaclnac~o BCG no Brasil~ ttospltal, Rio de Janeiro 27: 529-543, 1945. 17. Weill-ttall6, B.: 'La Vaccination Antituberculeuse par l ' I n j e c t i o n Sous-Cutande de BCG: Fresse todd. 36: 721-722, 1928.
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18. Wallgren, A.:
I n t r a d e r m a l Vaccinations W i t h ]3CG Virus, P r e l i m i n a r y Note, J. A.
M. A. 91: 1876-1881, 1928.
19. (a) Rosenthal, Sol Roy: Studies W i t h BOG: The P r e s e n t Method of BCG Cultivation and Vaccine Production P r a c t i c e d at the P a s t e u r /Institute and the Tiee Laboratories, Ant. Rev. Tubere. 35: 678-684~ 1937. (b) Rosenthal, Sol 1%y: The Multiple P u n c t u r e Method of BCG Vaccination, Am. Rev. Tubere. 39: 128, 1939. (e) Rosenthal, Sol Roy: L a V a c c i n a t i o n au BCG par la Methode des Piqures Superfieielles Multiples, Rev. de la tubere. 5: 815, 1939. 20. Rosenthal, Sol Roy: V a c c i n a t i o n au Moyen de UAntig~ne en Poudre (1)ess~ehd aprgs Congdlation) et de la P l a q u e t t e a Piqures Multiples, Ann. de l~Inst. P a s t e u r 75: 497, 1948. 21. N@gre, L., and Bretey, J.: V a c c i n a t i o n p a r le BCG par Scarifications Cutan6es~ Paris~ 1947, Masson & Cie. 22. F,irkhaug, K.: A Spring-Actuated ]~ultiple P u n c t u r e A p p a r a t u s for BOG Vaccination, Am. J. Clin. P a t h . 17: 751-754, 1947. 23. Rosenthal~ Sol Roy: Conservation du Baeille de Cahnette et Gugrin (BCG) par 1)essiccation aprhs Co~gdlation, Ann. de 1 'Inst. P a s t e u r 75: 209, 1948. 24. (a) Wallgren, A.: Calmette-Vaeeination in Sweden, Swedish N a t i o n a l Association A g a i n s t Tuhereulosis, 1947. (Pamphlet.) (b) l~ertzberg, G.: The A c h i e v e m e n t s of BCG Vaeeination~ Oslo, 29~8, J o h a n G r u n d t T a n u m Forlae. (Book.) (e) I{eimbeek, J.: BCG V a c c i n a t i o n in Nurses, Tubercle 29: 84, 1948. (d) Holm, J o h a n n e s : BCG Vaccination iu Denmark, Pub. H e a l t h Rep. 61: 1298-1316, 1946. 25. Boquet, A.~ and Negre, L.: Vaeeinatiolt P r e v e n t i v e Contre la Tuberculose par le BCG p a r A. Calmette~ C. Gu6rin, Paris, 1927, Masson et C~e. 26. (a) Aronson, J. D., and Palmer, C . E . : Experience W i t h BCG Vaccine in the Control of Tuberculosis Among N o r t h A m e r i c a n Indians, Pub. H e a l t h Rep. 61: 802-820, 1946. (b) Ferguson, R. G.: BCG V a c c i n a t i o n in Hospitals and S a n a t o r i a of Saskatchewan, Canad. J. Pub. H e a l t h 37: 435-451, 1946. 27. Sams, General C.: P e r s o n a l Communication. 28. Rosenthal, Sol Roy, Les]ie~ E. I.~ and Loewinsohn, E.: BCG V a c c i n a t i o n in All Age Groups: J. A. M. A. 136: 73-79, 1948. 29. Rosenthal, Sol Roy: I n Publication.