Comparative Experimental Study of Sensitivity to Tuberculin and

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Comparative Experimental Study of Sensitivity to Tuberculin
and Lepromin in Guinea-pigs Previously Inoculated with
BCG by Oral and Intradermal Routes
1_
R. D. Azulay and "de Kahn 3
Premunition by BCG for prophylaxis of
leprosy and tuberculosi s has been tri ed by
several routes of inoculation. Regarding th e
intradermal route, Brown et oZ. (] 1 )
showed that BCG protects against leprosy,
and D'Arey Hart et a7. ( 12) showed protection against tuberculosis.
In Brazil, oral BCG premunition has
b:'c n ca rri ed out in several million children
sin ce 1927. The value of the oral route with
respect to positive conversion of the tuberculin and lepromin tests has been
presented in several publications either in
th e field of leprosy ("I. 16 , 18) or in th e
fi eld of tuberculosis (1.R.15,17).
There has been discussion concernin g th e
immunologic and practical advantages of
one route over others (9, 10). This problem
is very important with respec t to costs of
administration and determination of th e
most practical u se of BCG premunition in
leprosy and tuberculosis in mass campaigns
in und erdeveloped countries.
A positive lepromin t est is regarded as a
sign of resistan ce against leprosy and a
positive tuberculin test is an indirect sign
indicating th e presence of some resistance
against tu bercu losis despite recognition of
the fact that tuberculin allergy and resistance against tuberculosis are different
phenom ena. Ncverth elcss, post-vaccin ation
all ergy is the onl y sign which p ermits evaluation , in children. of the effi ciency of
premunition. In elucidation of the problem
of th e optimum route of inoculation , an
investigation of th e lepromin and tubercu1 R ece ived for pllbli ca ti on 2'1 Ma rch 1969 .
~ Paper presented 10 Ih e I Xl h Internali on al Le p.
rosy Congress, Londo n , 16·2 1 Sepl embel' 1968.
3 R . D. AZlda y, M.D ., Chi ef of In st itllte of Lep·
rology, Nat ion al Service of Leprosy, Brazil , and
I'l'Ofe~sor of D e rm a l.o l o~ y a t U ni ve rsid ade Fed eral
F1l1minell se a nd at U ni versid ade do Estado da
G u a nab ara; H . Kahn , M .D .. Direc tor of Fllnd a((ao
Al:l llifo de P a iva. nCG Service. Brazil.
lin tests after oral and intraderm al inoculation of BCG in guin ea-pigs was undertaken. ( A similar study is in progress in new
born infants and children. The results will
be presented later.) This paper shows th e
6rst results of the experim ent; th e 6nal
results will be published after th e verifi cation of lepromin and tuberculin sensitization as evidenced by th e tests don e fi ve
months after BCG .inoculations.
MATERIALS AND METHODS
Materials. One hundred guin ea-p igs,
thrce to four months old, wcighin g approximately 350 gm. each, were used. Vaccinations were made with either lyophilized BCG produced by Glaxo Laboratories,
Greenford, England ( Batch B818 J) , or
BCG Moreau ( Rio de Janeiro strain ),
maintained altern atively in glycerinepotato and bile-potato media . .
Tuberculin hypersensiti vity was tes ted
with Old Tuberculin ( OT ) and PPD by
intradermal route. Lepromin hyp ersensiti vity was t ested with lepromin antigen,
Hayashi-Mitsuda type ( 16 X 107 bacilli per
ce. ).
Technic of vaccination. All the 100
guinea-pigs were negative to an initial tuberculin t est. They were di vided in four
groups (A, B, C and D ), each havin g 25
guinea-pigs.
Group A-Control group.
Group B-Intraderm al inoculation with
0.05 mg. of lyophilized BCG.
Group C- Intradermal inocul ation with
0.05 mg. of BCG Moreau ( Rio de J anei1'0
strain ) .
Group D - Oral inocul ation with 100 mg. of
BCG Moreau ( Rio de Janeiro strain ),
The inocula used were determined by
analogy with those used in newborn infants
373
1970
International Journal of Leprosy
374
'!.
100
90
L[ P
"'
".
z
<r
0-
10
0
8
REACTIVITY
F.I G.
POSITIVITY
1. Results of tes ts to tuberculin, PPD, and lepromin after vaccination with BCG .
Reacli vity : reactions having 1 or more mm. in diam eter. Positi vit y: reactions havin g 2
Dr more mm. in di ameter.
as recomm end ed by Glaxo Laboratories
and Viscondessa de Morais's Institute, Rio
de Jan eiro. Azu]ay et ol. (5-8) showed that
BCG oral vaccination in doses of 30 mg.
per kg. of animal weight induces 87 p er
cent lepromin positivity in guinea-pigs.
Amaral and Sorensen (2) obtained 90 per
cent positive tuberculin test and in newborn guinea-pigs after administration of 20
mg. BCG by mou th ( 13, H).
Thirty days after vaccination all guineapigs were t ested with OT, diluted to 1/ 10
units of PPD ( Ht 23-Copenhagen) and
TA BLE 1. Results uf tests to tuberculin,
P P D and lepromin after vaccination (cith
BGG.
R eactivity"
....'"
P PD
B(,G
('ontrol
BCG
%
%
%
%
22 .7
78 .7
39 .3
22
61
GLA)/:O INTRAOE.ANIC
-
G,.OUP C
l eG
UP
NOR[AU ' INTRAO[Mt IC
w
- - - TU'
......
- - .rpo
_
GROUP 0
L[I'
leG WOMau - OAAL
73.8
16 . 3
Lc~m:' ____ I--=~=- ~~~ _~ 5 ~~~
No. guin ca-pig,.;
BeG
-
Positi vityb
tl'ol
Tuberculin
, ---
~
- . - TU'
-----------('on-
GROUP 8
~
22
61
I
" H,eadivit y = reactions having
or more n1l11.
in diameter.
b Positivi ty = rea ctions hav ing 2 or more mm.
in diameter.
I
..~---+ -- .. -.. _- :
O "~
Z 3 .
- . -. "1'0
_
.
LEI'
FIC. 2. Sensitivity ,to tuberculin, PPD , and
lepromin in guinea-pigs previously inoculated
with BeG.
1
I
1
1
1
59 .1
27 .2
9.1
1
13 .6
27 .2
27 .2
1
1
59 .1
27.2
100.0
1
14 .2
100 .0
85 .0
25 .0
85 .7
1
76.1
28 .5
90.4
1
33 .3
85 .7
Group D. BCG Moreau oral
59 . 1
27 .2
81.8
I
1
Group C. BCG Moreau intradermal
63 .6
13 .6
4 .5
Group B. BCG Glaxo intradermal
1
22 .7
27.2
-
1
9.5
90.4
I.
76.1
-
50 .0
-
70 .2
9.1
-
-
-
1
1
1
1
21
Tuberculin
PPD
Lepromin
Tuberculin
PPD
Lepromin
Tuberculin
PPD
Lepromin
Tuberculin
PPD
Lepromin
Control
1
I
I
II
1
I
7
-
1
4 .5
-
Grou p A . Control
72
1
1
1
54.5
27.2
4 .5
1
I
54 .1
9.1
100 .0
1
80 .0
10 .0
85.0
1
76.1
14 .3
90.4
I
1
100 .0
-
14 .2
80.7
-
33 .3
I
I
7G. 1
90.4
-
50 .0
-
4 .5
.....
~
en
'"
~.
-d
>=l
(\)
S·
~
;;.
;;.
;3
d
""13
(\)
t"'<
G-
S-
.:
~
(\)
0'"
~
o
;i.:
<>:::
g.~
(\)
en
;:;-;::s
~
G-
<>:::
~
~
~
>I>-
-
-
w
yO?
-
-
1
1
1
1
1
21
-
9 .5
70 .2
-
-
-
14
Days
intrader~zal
13 .6
9 .1
27.2
Group D . BCG .1foreau oral
59 . 1
4.5
63 .5
Grou p C. BCG J[oreau
54 .5
13 .6
4 .5
Group B. BCG Glaxo intradermal
-
48
Hours
% Positivityb
PPD, and tuberculin, after BeG vaccination by different routes.
-
-
-
14
Days
to lepromin,
• Reactivity = reaction having 1 or m ore mm. in diameter.
b Positivi ty = reaction having 2 or more mm. in diameter.
Tuberculin
PPD
Lepromin
Tuberculin
PPD
Lepromin
Tuberculin
PPD
Lepromin
-
-
-
-
-
I
7
Group A . Control
Tuberculin
PPD
Lepromin
1
72
48
Hours
% Reactivitya
2. Sensitivity of guinea-pigs
Control
TABLE
376
I ntemational }ollmal of Leprosy
lepromin containing 16 X 107 bacilli/ ml.
by intradcrmal route. The readings "vere
mad e at 48 hours, a,ncJ at 7, 14 and 21 days.
Criteria for reading results. Positivity was
determined by the frequency of the largest
reactions measured in millimeters. A "reactive" test means any reaction independent
of th e size of it. The criteria of positivity
were as follows:
1. Positive tllJJercuIin test : reaction of 2
or more mm. at 48 hours.
2. ·Positive PPD test: reaction of 2 or
more mm . at 72 hours.
3. Positive lepromin test: reaction of 2
or more mm. at seven days.
RESULTS
Group A (control )
1970
Group D (oral BCG.Morcau).
1. Tuberculin test: 80%positive; thi s decreased to 76.1%, .33.3% and 9.5% at
the 72 hour, 7 day, and 14 day
readings respectively. Absence of any
reaction at 21st day reading.
2. PPD test: 14.3%positive but th e I'eae"
tivity was twi ce as hi gh (28.5%). Absencc of any reaction at 7, 14 and 21
days, respcctively.
3. Lepromin test: 85.0% positive; this
increased to 90.4% at th e 14th day
reading and dccreased to 76.1% at the
21st day readin g.
SUMMARY AND CONCLUSIONS
A definite sensitization of guinea·pigs to
Old
Tuberculin , PPD and lepromin was
1. Tuberc ulin test: absence of any reacinduced
by the adminis·tration of BCG. The
tion
2. PPD tes t: no positive test, but some reactivity reactions were a little bit higher
discrete reactivity ( 1 mm. ) was than the positivity reaction s.
The hi ghest points of the curves of posipresent in 22.7%at seven days.
3. Lepromin test: 4.5% positive test and tivity were: 48· hours for th e tuberculin
test, 72 hours for th e PPD test and seven
27.2%reactive tes t at seven days.
days for th e lepromin test.
Group B (intradermal BCG.Glaxo).
The lepromin reaotion in guinea·pigs is
of
shorter duration than in human beings.
1. Tuberculin test: 54.5% positive and
The induced sensitization of th e guinea63.6% reactive test. It is interestin g to
observe that 13.6%of the positive tests pigs varied according to the type and route
were still evident at th e seventh day. of administration of BCG.
At the later readings all reaction s had
High est tuberculin sensitization was
disappeared.
achieved in Group D (oral BCG-Moreau ).
2. P<PD test: 27.2% positive; 9.1% rePPD sensitization was high est in Group
mained positive until the seventh day. B (intradermal BCG-Glaxo).
3. Lepromin test: 27.2% positive; 4.5%
Lepromin sensitization was very low in
remained positive until the 21st day.
Group B ( intradermal BCG-Glaxo ); it was
very high in Group D ( Oral BCG Moreau )
Group C (intradermal BCG·Moreau).
and th e hi ghest in Croup C ( intradermal
1. Tuberculin test : 59.1% positive; 14.2% BCG-Moreau) .
remained positive until the seventh
In Groups C and D , which received
day; at later readings all reaction had BCG-Moreau, the lepromin reactions were
disappeared.
more p ersistent; in the Group D ( oral BCG
2. PPD test: 9.1% positive, but the reac- Moreau ) the reactions were even more
tivity was three tim es higher (27.2%); persistent than in th e Group C ( intraderabsence of any reaction at later read- mal BCG-Moreau).
ings.
As th e oral administration of BCG is
3. Lepromin test: 100%positive; this de- easier and cheaper it should be recomcreased to 70.2% and to 50% at the mended in the under developed countries,
14th and 21st day readings, respec- in the prophylaxis of leprosy and tubercutively.
losis.
38,4
Azulay & Kahn: Sensitivity to Tuberculin & Lepromin in Guinea-pigs
RESUMEN
La administracion de BCG indujo en
cobayos un a sensibilizacion bi en definida a
tube rculina antigu a, PDD y lepromin a. Las
reacciones de re ac tividad fucron ligeramente
mayo res que las reacciones de positividad .
Los puntos mas altos de las curvas de
positivid ad fueron : 48 horas para la prueba
de tuberculin a, 72 ho ras para la prueba con
PPD y siete dias pa ra la pru eba con 1epromina.
La reaccion de lepromin a en co bayos es de
menor duracion que en seres hum anos.
La sensibilizac ion que se indujo en los
cobayos vario de ac uerdo co n los tipos de BCG
utilizados y vias de administracion .
La mayo r sensibili zacion contra la tuberculin a se obtuva en el Grupo D (BCG oralMoreau).
La sensibilizacion contra PPD fue mayor
en el Grupo B (BCG intradermico- Glaxo) .
La sensibilizacion contra la lepro min a fue
muy baja en el Grupo B (BCG intradermicoGlaxo ); mu y alta en el Grupo D ( BCG oral
- Moreau) y mayo r de tod as en el Grupo C
(BCG intrade rmico- Moreau) .
En los Grupos C y D , que recibieron BCG
-Moreau, las reaccion es de lepromin a fueron
mas persistentes; en el Grupo D (BCG oralMoreau) las reacciones fueron allll mas persistentes que en el Grupo C (BCG intradermico
- Moreau).
Ya que la administracion oral de BCG es
mas facil y de menor costo, debe se r recomendada para paises en desa rrollo, para la profilaxis de la lepra y la tuberculosis.
377
La sensibilisation par Ie PPD s'est revelee
la plus elevee dans Ie groupe B (BCG intradermique Gl axo).
La sensibilisation par la lepromine etait tres
fa ible dans Ie groupe B (BCG intradermique
Glaxo); elle etait tres elevee d ans Ie groupe D
(BCG Oral Moreau ) et la plus eleves dans Ie
gro upe C (BCG intradermique Moreau) .
Les reaGtions a la le promine sont apparues
les plus persistantes dans les groupes C et D
qui ava ient recru du BCG Moreau ; pour Ie
groupe D (BCG oral Morea u ); les reaction s
eta ient meme encore plus persistantes que dans
Ie groupe C. (BCG intradermique Moreau ) .
L'administration de BCG ora l dev rait etre
recomm andee pour la prophylaxie de la lepre
et de la tuberculose dans les pays en voie de
developpement, vu qu'elle est plu s facile et
plus economique.
REFERENCES
1. ALvIl"r, A. C. and NASCIJ\IENTO, E. A. D ez
anos de vaci nac;:ao BCG concorrente na
Favela d a Praia d o Pinto. J. Brasileira
Doene. T onie. 2 ( 1966 ) 8-9.
2. A",rARAL, J. P. an d SOERENSEN, B. BCG
pol' via oral em eob aios. 0 H ospital 58
( 1960 ) 1053-1057.
3. ASSIS, A. PASSADO, presente e futuro da
vaeinac;:ao BCG. Rev. Servo Nae. Tubere.
8 (1964) 7-30.
4. AZULAY, R. D . A aean do BCG sobre a
reac;:ao le promini ea. 0
H ospital , 34
( 1948) 853-856.
5. AZULAY, R. D . Our exp eri ence w ith the
leprom in ·t est from 1944 to 1955. II Congo
RESUME
Intern . Alergia, Rio de Janerio, 1955.
L'administration de BCG a entraine une
6. AZULAY, R. D. and AZ ULAY, J. D. A virasensibilisation indi scutable de cobayes a la
gem da lepromin o-readio pela adminisVieille Tuberculine, au PPD et a la lepromine .
trac;:ao do BCG. 1301. Servo Nae. Lepra 12
Les reactions de r eac tivation etaient
(1953) 310-312.
legerement plus prononcees que les reactions
7. AZ ULAY, R. D. and NEVES, R. G. Comde positivite.
p ortamento do teste leprominieo em
Les points les plus eleves des courbes de
eobaios beeegeizados p Ol' via oral. Rev.
positivite obtenues etaient les suiv ants; 48
brasileira L epro!. 2 6 ( 1958 ) 103-106.
heures pour I'epreuve a la tuberculine, 72
8. AZULAY, R. D ., NEVES, R. G. and Az ULAY
heures pour I'epreuve au PPD et 7 jours pour
.T . D . A reac;:ao le prominica apos previ~
I'epreuve a la lepromine .
in oculac;:ao do BCG e de Mycobacterium
La reaction a la lepromine chez les cobayes
tllberculosis morto p OI' irradia<;ao. Rev.
temoignait d' une duree plus cOllrte que chez les .
brasileira Lepro!. 27 ( 1959) 81-86.
etres hllmains .
9. BLASI, A. and CuRCI, G. Investigac;:oes exLa sensibilisation ainsi indllite chez les
p erimentais sabre a vacinac;:ao com BCG
cobayes variait en fonction du type de BCG
pOI' vi a oral segundo A. de Assis. 0 Hoset de son mode d'administration.
pita l 47 ( 1955) 215-229.
La sensib ilisation la plus forte a la tubercu10. BOTES, V., PARASCHJVESCO, M ., ANGELESline a ete obtenue dans Ie groupe D (BCG
CO, 1. , BONCIU, C. and PETROVICI, M.
Moreau Oral) .
International l ou'm al of L eprosy
378
E studo exp e rimental sabre a vaci na9ao
BCG intensiva pro via oral. 0 H ospital 61
(1962 ) 441-45Q.
11. BROWN, J. A. K., STONE, M. M. and SUTHERLAND, I. B.C .G. vaccination of children
again st leprosy in U ganda. Results a t end
of second follow up. Brit. Med . J. 1
(1968) 24-27.
12. H ART, P . D 'A. , SUTHERLAND, I. and
THOMAS, J. The immunity confe rred b y
effecti ve BCG and vole b acillius vaccines,
in relation to individu al va riations in in'duced tube rculin sensitivity and to tech nical variations in the vaccin es . Tubercle
48 ( 1967 ) 201-210.
13. JESPERSEN, A. and BENTZON, M. W. Rela-
tionship b e tween tuber culin sensitivity
and acquired resistan ce in guin ea-pigs
vaccin ated with BCG strains of different
virulence. Ac ta Pa th . Microbiol. Scandinav. 71 ( 1967 ) 114-131.
14. JESPERSEN, A. , BENTZON, M. W . and
MAGNUSSON, M. Acquired resistance to
tuberculos is and tuberculin sensitivity in
1970
g uin ea-pigs vaccina ted w ith a small or a
large d ose of BCG vacci ne. Acta P a th.
Microbiol. Scandinav. 56 (1962 ) 193-218.
15. ROSEMBERG, J. Considera90es sobre 0 BCG
liofili zado. 0 H ospital 67 (1965) 215-
237.
16. ROSE}'fBERC, J., SOUZA CAMPOS, N. and
A UN, J . N. Rea9ao de Mitsuda induzida
p OI' efcito d e di ve rsos esquemas d e vacina9ao BCG oral e p ela tecnica de multipuntures de Rosenth al. Rev. brasileira
L ep ro l. 20 (1952 ) 183-196.
17. SrCNORINI, L. F . Esperie nze di vaccinazione antituhercol are dell a vacia pe r via
orale. Lo Sperimentale 106 (1 ) (1956 )
88-100, and 0 H ospital 50 (2 ) ( 1956 )
161-162.
18. SOUZA CAMPOS, N ., L ESER, 'vV ., QUAGLIATO, R. , BECHELLI, L. M., and ROTBERG,
A. Viragem da lepromino-rea9ao em
fun 9ao de diferentes es timulos. Influencia
da idad e, nessa v irage m, no grupo etario
de 6 a 43 meses. Rev. brasil eira L eprol .
29 (1962 ) 3-20.