- Email: [email protected]
Mycobacterium tuberculosis tuberculin devoid of BCG common antigens
Zbl. Bakt. Hyg. , 1.Abt. Orig. A 251, 399-401 (1982)
Cantacuzino Institute, Bucharest, Roum ania Clinical Institute of Phtisiology, Bucharest, Rouman ia
1
Mycobacterium tuberculosis Tuberculin Devoid of BCG Common Antigens M. tuberculosis Tuberkulin ohne kreuzreagierende BCG-Antigene
D . STAVRI, HENRIETTE STAVRI, 1. CLAICIU\ and DANA NICULESCU
With 1 Figure' Received December 23, 1981
Zusammenfassung Auch die heutigen sog. gereinigten Kulrurfiltrate von M ykobakterien enthalten neben spezies-spezifischen Antigenen noch zahlreiche mit anderen Spezies kreuzreagierende Antigene. Das behindert die Tuberkulindiagnostik besonders in den Landern , in denen noch die generalisierte BCG-Impfung dur chgefiihrt wird, da hier die diagno stische Mogl ichkeir einer virulenten Superinfektion mit M . tuber culosis praktisch entf allt, Mirtels der praparariven Gegenstrom-Immunelektrophorese wurde eine verbesserte Reinigung von Kultur filtraten des Stammes H37Rv angestrebt. Dabei wurden nur Antigene gepoolt, die mit Antiseren des Stammes H37Rv reagierten, In Tierversuchen reagierten im Hauttest mit diesem PPD-A ausschlieBlich Meerschwe inchen positiv, die mit M . tub erculosis infiziert waren, jedoch nicht die mit dem rurnanischen BCG-Tochterstamm infizierren Tiere . Ober er ste begrenzre klinische Pilotsrudien, die noch nicht voll befriedigcn konnren , wird berichtet.
The culture filtrates of Mycobacteria contain a mixture of antigens very different as concerning the chemical composition and the eliciting activity of tuberculin allergy. Some of these antigens are specific for mycobacterial species to which they belong and others are shared with various mycobacterial species. The common antigens induce cross-reactions with different tuberculin derivatives, which obscure the clinical diagnosis and limit the significance of tuberculin skin testing as an aid for the diagnosis of tuberculosis. In human and veterinary clinics , in tuberculosis epidemiology, there is a great need to obtain specific purified tuberculins (Daniel, 1976). A review concerning th e iso lation of mycobacterial a nti gens was made by Daniel and Janicki (1978). In our laboratory, a new sepa ratio n merhod, the preparative countercurrent immunoelectrophoresis had been elaborated in order to obtain from unheated culture filtrates of Mycobacterium tub erculosis, stra in H3 7Rv a protein purified derivative
400
D. Stavri, H. Stavri, I. Claiciu, and D. Niculescu
deprived of the common antigens with the roumanian BCG substrain (Stavri et al., 1980). Briefly, three agarose layers were pulled in a preparative electrophoresis apparatus column. The bottom layer consists of a mixture of BCG anti-immunoglobulins, the middle layer of 6% agarose and the top layer of a mixture of agarose with antigens from unheated culture filtrate of Mycobacterium tuberculosis, strain H37Rv. The electrophoresis was carried out with the cathode on the top of the column. The elution was carried out by using a peristaltic pump and the fractions were automatically collected. The distribution of the antigens in the fractions was determined by fussed rocket immunoelectrophoresis. Only the fractions containing the antigens which reacted with Mycobacterium tuberculosis, strain H37Rv antiserum were pooled. The pooled fractions, designated PPD A, were bioequivalated with 2 UT PPD and were tested on two groups of 15 guinea pigs, previously BeG vaccinated or infected with Mycobacterium tuberculosis, in order to establish its specificity in eliciting the tuberculin allergy. PPD A revealed the tuberculin allergy only in the guinea pigs infected with Mycobacterium tuberculosis. These results obtained with animals justified further human trials to confirm the specificity of our preparation. PPO A was bioequivalated again on man with 2 UT PPO and was administered simultaneously with the reference PPO on 100 children, 6-9 months after BCG vac-
'0
«
c,
0
Q"g CL 0...
0
~
V>
""D
0.5
9CL QCL
<>:
9
j
-s,
0 -3
- - --.:-- - - - - - - ----- ~- X
Post vaccin a l scc rr emm)
,,
,
- --
'
"
-, -- ~ ,--
,,
"z
-,
, 5
, -,
,,
,
- - - - - - -- ~ ,
,,
,
12
36
Time after
BeGvoccmohon (mon t hs)
Fig. 1. The correlation between the report on the dimension of the reactions to PPD A and 2 UT PPD standard (y), time after vaccination (x) and dimensions of post-vaccinal scar (z).
Mycobacterium tuberculosis Tuberculin
401
cination at birth. These children had not been in contact with tuberculous people and had a post-vaccinal scar more than 3 mm. It can be established that with the PPD A the induration diameter did not reach the positivity limit, while using the reference product 13.26% of the reactions exceeded this limit (Claiciu et al., 1980). Our experiments in a limited field trial (280 children aged 7-18 years) demonstrated that the specificity of the PPD A was expressed only in the case of simultaneous existence of the post-vaccinal and post-natural infection allergies (Fig. 1). Our preliminary results prove that the post-vaccinal allergy is directly proportional to the post-vaccinal scar dimension and inversely proportional with the period of time between the vaccination and the allergy determination. These results represent a confirmation of the present knowledge concerning the relationships among these three sizes. In conclusion, the possibility to obtain a specific tuberculin product opens the outlook to eliminate the difficulties arising in diagnosis and to set up a useful tool in the epidemiologic survey of tuberculosis in the areas where systematic BeG vaccination is still performed. The results obtained here do not satisfy us in totality. But studies to improve the methods for the isolation of specific antigens are in progress.
References Claiciu, I., D. Stavri, R. Biirbulescu, G. Algeorge, Beatrice Calciu, M. Calciu, Henriette Stauri, and Dana Niculescu: The specificity of a tuberculin devoid from cross-reacting
antigens common with BCGin elicitingthe delayedhypersensitivity in men. Arch. Roum. Path. Exp. Microbiol. 39 (1980) 369 Daniel, T. M.: Tuberculin antigens. The need for purification. Amer. Rev. resp. Dis. 133 (1976) 717
Daniel, T. M. and B. W.Janicki: Mycobacterial antigens: a review of their isolation,
chemistry and immunological properties. Microbiol. Rev. 42 (1978) 84 Stauri, D., Henriette Stavri, Dana Niculescu, G. Algeorge, and Maria Stoian: Preparative
countercurrent immunoelectrophoresis. Application at the separation of specific tuberculoproteins. Rev. Roum. Biochem. 17 (1980) 217 Dipl.-Chem. Dinu Stauri, Institutul Cantacuzino, Splaiul Independentei 103, 7620 Bucuresti, 35, Romania
Cantacuzino Institute, Bucharest, Roum ania Clinical Institute of Phtisiology, Bucharest, Rouman ia
1
Mycobacterium tuberculosis Tuberculin Devoid of BCG Common Antigens M. tuberculosis Tuberkulin ohne kreuzreagierende BCG-Antigene
D . STAVRI, HENRIETTE STAVRI, 1. CLAICIU\ and DANA NICULESCU
With 1 Figure' Received December 23, 1981
Zusammenfassung Auch die heutigen sog. gereinigten Kulrurfiltrate von M ykobakterien enthalten neben spezies-spezifischen Antigenen noch zahlreiche mit anderen Spezies kreuzreagierende Antigene. Das behindert die Tuberkulindiagnostik besonders in den Landern , in denen noch die generalisierte BCG-Impfung dur chgefiihrt wird, da hier die diagno stische Mogl ichkeir einer virulenten Superinfektion mit M . tuber culosis praktisch entf allt, Mirtels der praparariven Gegenstrom-Immunelektrophorese wurde eine verbesserte Reinigung von Kultur filtraten des Stammes H37Rv angestrebt. Dabei wurden nur Antigene gepoolt, die mit Antiseren des Stammes H37Rv reagierten, In Tierversuchen reagierten im Hauttest mit diesem PPD-A ausschlieBlich Meerschwe inchen positiv, die mit M . tub erculosis infiziert waren, jedoch nicht die mit dem rurnanischen BCG-Tochterstamm infizierren Tiere . Ober er ste begrenzre klinische Pilotsrudien, die noch nicht voll befriedigcn konnren , wird berichtet.
The culture filtrates of Mycobacteria contain a mixture of antigens very different as concerning the chemical composition and the eliciting activity of tuberculin allergy. Some of these antigens are specific for mycobacterial species to which they belong and others are shared with various mycobacterial species. The common antigens induce cross-reactions with different tuberculin derivatives, which obscure the clinical diagnosis and limit the significance of tuberculin skin testing as an aid for the diagnosis of tuberculosis. In human and veterinary clinics , in tuberculosis epidemiology, there is a great need to obtain specific purified tuberculins (Daniel, 1976). A review concerning th e iso lation of mycobacterial a nti gens was made by Daniel and Janicki (1978). In our laboratory, a new sepa ratio n merhod, the preparative countercurrent immunoelectrophoresis had been elaborated in order to obtain from unheated culture filtrates of Mycobacterium tub erculosis, stra in H3 7Rv a protein purified derivative
400
D. Stavri, H. Stavri, I. Claiciu, and D. Niculescu
deprived of the common antigens with the roumanian BCG substrain (Stavri et al., 1980). Briefly, three agarose layers were pulled in a preparative electrophoresis apparatus column. The bottom layer consists of a mixture of BCG anti-immunoglobulins, the middle layer of 6% agarose and the top layer of a mixture of agarose with antigens from unheated culture filtrate of Mycobacterium tuberculosis, strain H37Rv. The electrophoresis was carried out with the cathode on the top of the column. The elution was carried out by using a peristaltic pump and the fractions were automatically collected. The distribution of the antigens in the fractions was determined by fussed rocket immunoelectrophoresis. Only the fractions containing the antigens which reacted with Mycobacterium tuberculosis, strain H37Rv antiserum were pooled. The pooled fractions, designated PPD A, were bioequivalated with 2 UT PPD and were tested on two groups of 15 guinea pigs, previously BeG vaccinated or infected with Mycobacterium tuberculosis, in order to establish its specificity in eliciting the tuberculin allergy. PPD A revealed the tuberculin allergy only in the guinea pigs infected with Mycobacterium tuberculosis. These results obtained with animals justified further human trials to confirm the specificity of our preparation. PPO A was bioequivalated again on man with 2 UT PPO and was administered simultaneously with the reference PPO on 100 children, 6-9 months after BCG vac-
'0
«
c,
0
Q"g CL 0...
0
~
V>
""D
0.5
9CL QCL
<>:
9
j
-s,
0 -3
- - --.:-- - - - - - - ----- ~- X
Post vaccin a l scc rr emm)
,,
,
- --
'
"
-, -- ~ ,--
,,
"z
-,
, 5
, -,
,,
,
- - - - - - -- ~ ,
,,
,
12
36
Time after
BeGvoccmohon (mon t hs)
Fig. 1. The correlation between the report on the dimension of the reactions to PPD A and 2 UT PPD standard (y), time after vaccination (x) and dimensions of post-vaccinal scar (z).
Mycobacterium tuberculosis Tuberculin
401
cination at birth. These children had not been in contact with tuberculous people and had a post-vaccinal scar more than 3 mm. It can be established that with the PPD A the induration diameter did not reach the positivity limit, while using the reference product 13.26% of the reactions exceeded this limit (Claiciu et al., 1980). Our experiments in a limited field trial (280 children aged 7-18 years) demonstrated that the specificity of the PPD A was expressed only in the case of simultaneous existence of the post-vaccinal and post-natural infection allergies (Fig. 1). Our preliminary results prove that the post-vaccinal allergy is directly proportional to the post-vaccinal scar dimension and inversely proportional with the period of time between the vaccination and the allergy determination. These results represent a confirmation of the present knowledge concerning the relationships among these three sizes. In conclusion, the possibility to obtain a specific tuberculin product opens the outlook to eliminate the difficulties arising in diagnosis and to set up a useful tool in the epidemiologic survey of tuberculosis in the areas where systematic BeG vaccination is still performed. The results obtained here do not satisfy us in totality. But studies to improve the methods for the isolation of specific antigens are in progress.
References Claiciu, I., D. Stavri, R. Biirbulescu, G. Algeorge, Beatrice Calciu, M. Calciu, Henriette Stauri, and Dana Niculescu: The specificity of a tuberculin devoid from cross-reacting
antigens common with BCGin elicitingthe delayedhypersensitivity in men. Arch. Roum. Path. Exp. Microbiol. 39 (1980) 369 Daniel, T. M.: Tuberculin antigens. The need for purification. Amer. Rev. resp. Dis. 133 (1976) 717
Daniel, T. M. and B. W.Janicki: Mycobacterial antigens: a review of their isolation,
chemistry and immunological properties. Microbiol. Rev. 42 (1978) 84 Stauri, D., Henriette Stavri, Dana Niculescu, G. Algeorge, and Maria Stoian: Preparative
countercurrent immunoelectrophoresis. Application at the separation of specific tuberculoproteins. Rev. Roum. Biochem. 17 (1980) 217 Dipl.-Chem. Dinu Stauri, Institutul Cantacuzino, Splaiul Independentei 103, 7620 Bucuresti, 35, Romania