Protective effect of BCG in Ahmednagar, India

Tubercle (1987) 08, 0 Longman Group

169-176 UK Ltd. 1987

PROTECTIVE

EFFECT

OF BCG

IN AHMEDNAGAR,

INDIA

J. L. Stanford, N. Sheikh,” G. Bogle, C. Baker, H. Series, and P. Mayo School

of Pathology, Middlesex Hospital Medical School, and ?? Evangelize Booth Hospital, Ahmednagar

Riding House Street, 414001, Maharashtra,

London lndia

W7P

7LD

Summary As part of a series of investigations to determine the effect of sensitisation by environmental mycobacteria on the efficacy of BCG vaccination in India, this study was carried out in Ahmednagar in Maharashtra. A preliminary skin test survey showed that the rate of sensitisation with age was much lower than in Agra, the site of a previous study, and BCG vaccination scars were associated with considerable enhancement in sensitisation to Tuberculin and other reagents. It was possible to set up prospective BCG vaccination studies in pre-school and primary and secondary school children. Follow up with skin tests were carried out 1 and 2 years later. By the second year, results were obtained almost identical with those 10 years after BCG administration in the UK. On this basis it is proposed that the vaccine is likely to provide a considerable level of protection in Ahmednagar. The results of this study also resemble those obtained in the very youngest age group studied in Agra. The marked differences between Indian towns strongly suggest the influence of exposure to mycobacteria in the environment. R&urn6 Cette 6tude. men6e 81 Ahmednagar, Maharashtra, fait partie d’une sbrie de recherches destinbes B determiner I’effet de la sensibilisation par les mycobact&ies de I’environnement sur I’efficacitb de la vaccination BCG en Inde. Une enquQte pr6liminaire par tests cutan& avait montr6 que I’augmentation de la sensibilisation avec I’6ge dtait beaucoup plus faible qu’6 Agra, le lieu air une 6tude antbrieure avait BtB r6alis6e. et que la pr6sence de cicatrice de vaccination BCG dtait associbe B une augmentation consid&able de la sensibilisation B la tuberculine et autres produits. II a 6t6 possible de rbaliser des Etudes prospectives concernant la vaccination BCG chez des enfants avant I’lge d’entree 6 I’&ole, et B I’bge de I’enseignement primaire et secondaire. Un suivi a 6th effectu6 par des tests cutan6s 1 an et 2 ans plus tard. Les resultats obtenus 2 ans apr&s ont 6t6 semblables g ceux obtenus 10 ans apr6s I’administration de BCG au Royaume-Uni. Sur cette base, les auteurs suggerent qu’apparemment la vaccination BCG conf&e un niveau consid&able de protection 8 Ahmednagar. Les rbsultats de cette dtude sont comparables 6galement & ceux obtenus dans un groupe d’enfants beaucoup plus jeunes, 6tudi6s B Agra. Les diff6rences marquees trouv6es entre les diverses villes de I’lnde suggerent fortement I’influence de I’exposition aux mycobactt5ries de I’environnement. Resumen Este estudio efectuado en Ahmednagar, Maharashtra, forma patte de una serie de investigaciones destinadas a determinar el efecto de la sensibilizacidn por micobacterias ambientales sobre la eficacia de la vacunaci6n BCG en India. Una

170

Stanford

and others

encuesta preliminar con pruebas cutaneas habia mostrado que el aumento de la sensibilizacion con la edad era mucho m&s bajo que en Agra, lugar donde se habia realizado un estudio anterior, y que la presencia de cicatriz de vacunacion BCG estaba asociada a un aumento considerable de sensibilizacion a la Tuberculina y a otros productos. Se tuvo la posibilidad de realizar estudios prospectivos con respect0 a la vacunacion BCG en nines preescolares, y de la enserianza primaria y secundaria. Se efectuo un seguimiento con pruebas cutaneas un ario y dos arias mas tarde. Los resultados obtenidos al cabo de 2 atios fueron similares a aquellos obtenidos 10 anos despues de la vacunacion de BCG en el Reino Unido. En base a estos resultados se sugiere que la vacunacion BCG aparentemente confrere un nivel considerable de protection en Ahmednagar. Los resultados de este estudio son comparables igualmente a 10s obtenidos en un grupo de nines de mucho menos edad estudiados en Agra. Las diferencias importantes encontradas entre las diversas ciudades de India sugieren fuertemente la infleuncia de la exposition a las microbacterias ambientales. Introduction Considerable doubt exists as to whether BCG has any protective value against either tuberculosis or leprosy in India. This follows the first reports of the joint lCMR/WHO trial of BCG against tuberculosis in South India [I, 21 which showed no protection. However, a retrospective study of protection against leprosy achieved in the same trial was much more promising, and a level of protection against tuberculosis is being revealed with time [3]. Nonetheless, the protective effect has proved smaller than was hoped. The most plausable explanation lies in an effect of the environment, in particular of the mycobacteria in the environment 141, although opinions differ as to how this might work. In our previous study carried out among young children living in Agra [5], and in the present study, we have sought to investigate the effects of different mycobacterial environments on small prospective studies of BCG vaccination. It is not possible to measure protective effects directly with such small studies. Thus, an immunological parametermultiple skin testing-has been used in an attempt to assess protection indirectly. The assumption has been made that the results obtained in the UK after BCG represent the protective immune state, and the results of our studies are compared with this. In Agra the majority of school children were already Tuberculin positive whether they had BCG scars or not. Thus pre-school children were vaccinated and re-skin tested 1 and 2 years later. It was found that only the youngest children (mean age 1.2 years) produced reactions at the 2-year follow up equivalent to the results in the UK 10 years after BCG, whereas the reactions of the slightly older children (mean age 4.2 years) were larger. This increased size was thought to be due to sensitisation by environmental mycobacteria not reflected by the pre-vaccination skin tests. In support of this, a very high level of sensitisation to many mycobacterial species was found amongst non-BCG vaccinated older children. Ahmednagar, which is 200 k east of Bombay in Maharashtra, India, was selected for the second part of our study on the basis of a preliminary multiple skin test survey of school children described below. This showed that only 15.1 % of children aged 5-10 years, and 36.8 % of children aged 11-15 years without BCG scars were Tuberculin positive. Similar children in Agra were 65.1 % and 81.6 % Tuberculin positive respectively 151. Responsiveness to other mycobacterial reagents was also much lower than was found in Agra. Thus, in Ahmednagar it was possible to BCG vaccinate a reasonable proportion of school children as well as pre-school children. An account is given of skin test studies before and 1 and 2 years after BCG vaccination.

BCG in Ahmednagar

171

Materials and methods The reagents The skin test reagents used were new tuberculins as described before j5,6.71. The reagents were administered as intradermal injections of 0.1 ml in the volar surfaces of the forearms, and diameters of induration were measured after 72 h. In general, induration with a mean diameter of 2 mm or more was taken as a positive response, as in our previous studies. The reagents used in the preliminary survey were Tuberculin, Aviumins A, B, and C 181, Scrofulin, Kansasin, Marinin, Gordonin, Leprosin A and Vaccin. These were prepared from Mycobacterium tuberculosis, M. avium types A and B, M. intracellulare (Aviumin C], M. scrofulaceum, M. kansasii, M. marinum, M. gordonae, M. leprae and M.vaccae respectively. The pre-vaccination selection test reagents were Tuberculin and Vaccin for the pre-school children, and Tuberculin with three groups of three reagents for the older children. One group of reagents comprised the Aviumins A, B, and C; the second group comprised Flavescin, Rhodesin and Chitin prepared from M. flavescens, M. rhodesiae and M. chitae respectively. The last group consisted of Vaccin, Diernhoferin and Ranin 2, the two latter reagents being prepared from M. diemhoferi and M. fortuitum type II (giae or peregrinum) respectively. The concentrations of the reagents were 20 pg protein per ml for Vaccin, 10 pg/ml for Leprosin A, the 2 pg/ml for each of the other reagents. The first year follow-up tests were carried out with the same reagents in each child as were used in the selection tests, but the second year follow-up was carried out with Tuberculin and Leprosin A in the pre-school children, and with Tuberculin, Scrofulin, Leprosin A and Vaccin in the school children. The vaccine used was the standard recommended dose of Glaxo freeze-dried BCG, kindly supplied free of charge by the manufacturers. It was given by intradermal injection into the upper left deltoid region.

The preliminary

survey

This was carried out (by G.B.] in 1981 on 357 children attending schools in Ahmednagar, using Tuberculin and three combinations of three other reagents: 191 children were aged between 5 and 10 years, and only 19 of them had BCG scars. Of the 166 children aged 1 l-l 5, 54 had BCG scars. For simplicity, the results for this preliminary study are combined with the data collected when children were being selected for vaccination (by C.B.) in 1982, and with data collected from further children at the time of the second year follow-up (by P.M.) in 1984.

The prospective

vaccine study

A total of 674 children were screened for suitability for BCG vaccination. The vaccine was given to all those with reactions to Tuberculin of less than 5 mm who did not have a scar of previous BCG vaccination. These were the same selective criteria as were used in our studies in Agra [5] and the Lebanon [9]. Forty children of pre-school age attending Wakedi Creche were studied: 34 were suitable for vaccination and 32 were vaccinated; 6 already had BCG scars. Of 361 schoolchildren aged 5-10 years, 199 had BCG scars and 29 of those without scars were Tuberculin positive. Of 273 children aged 1 l-l 5 years, 180 had BCG scars and 41 of those without scars were Tuberculin positive. Thus 133 of the younger children and 52 of the older children were suitable for BCG vaccination, and the great majority received it.

The follow-up One and two years after vaccination

as many as possible of the children

were followed

up

172

Stanford

and others

with repeat skin testing, and examined for the scar of the BCG we had given. In the second follow-up a number of children not in the study were also tested and given BCG if they needed it.

Results The initial

survey

and pre-vaccination

results

The results are shown in Table I for each of the age groups studied. It can be seen that the rate of acquisition of Tuberculin positivity in the non-vaccinated children increases with age from 2.9 %/3.4 years, i.e. 0.9 % per year for the pre-school children to (17.5-2.9 )%/(8.4-3.4) years i.e. 2.9 % per year for the 8-10 year olds to (40.5-17.5)%/(12.8-8.4) year i.e. 5.8 % per year for the older children (see Fig. 1). With the exception of Aviumin B, Gordonin and Rhodesin which probably represent species absent in Ahmednagar, there is a similar increase in positivity with age to most reagents. BCG significantly enhanced positivity to Tuberculin in all age groups, and it also enhanced positivity to each of the other reagents tested in reasonable numbers of children, excepting those made from species absent from the environment. Of the six pre-school children with BCG scars (omitted from Table I), four were tuberculin positive with a mean reaction size of 8.3f2.4 mm. Results

of the follow-ups

Among the children of Wakedi Creche 34 had been given BCG, and of these 32 were followed-up one year after vaccination, and 23 were followed-up in the second year. Of the 131 children aged 5-10 years who were vaccinated, 125 were followed up in the first year and 53 were followed up in the second year. Of the 48 children vaccinated in the older age group, 47 were tested in the first follow-up and 20 in the second. The results obtained are shown in Table II, and those for Tuberculin are shown in Figure 1.

Table I. The results of initial skin tests from the preliminary selection procedure. No KG

Tuberculin Mean +ve size Aviumin A Aviumin 6 Aviumin C Scrofulin Kansasin Marinin Gordonin Vaccin Leprosin A Flavescin Rhodesin Chitin Ranin 2 Diernhoferin

scar

survey and from the vaccination

BCG scar present

t6

61-O

77+

610

11+

(3.4 yl

(8.4 yl

(12.8 yl

18.3 yl

(12.7 yl

l/34 2.9 % 2mm -

611349 17.5 % 10.1 f4.5 mm 15/108 13.9 % 7.1 % 7199 28194 29.8 % 717 1 9.9 % IO/51 19.6 % 8l51 15.7 % 4.6 % 3165 IO/95 10.5 % 8/80 10.0 % 4l94 4.3 % 1.1 % 1194 4.3 % 4794 2124 8.3 % 4.2 % 1124

1171289 11.5k5.0 16167 5/86 31176 291131 6/37 9137 l/28 30/148 32/l 12 8137 1I37 19137 407 2117

0040%

-

40.5 % mm 23.9 % 5.8 % 40.8 % 22.1 % 16.2 % 24.3 % 3.6 % 20.3 % 28.6 % 21.6% 2.7 % 51.4 % 23.5 % 11.8 %

1171242 9.7k4.3 271101 13/101 51/99 5/31 2l5 l/5 l/7 457124 12131 3122 0122 3122 12/93 1 l/93

48.3 % mm 26.7 % 12.9 % 51.5 % 16.1 % 40.0 % 20.0 % 14.3 % 36.3 % 38.7 % 13.6 % 0 % 13.6 % 12.9 % 11.8 %

181/301 10.8k5.1 25169 5l78 33175 34789 2119 4/19 l/l3 56/150 46/80 IO/60 2l60 30/60 9/61 lo/61

60.1 % mm 36.2 % 6.4 % 44.0 % 38.2 % 10.5 % 21.1 % 7.7 % 37.3 % 57.5 % 16.7 % 3.3 % 50.0 % 14.8 % 16.4 %

BCG in Ahmednagar

173

80

o/o 60

40

20

3.4

8.4

12.8

years

of

age

Figure I. The increases in Tuberculin positivity with age and following BCG vaccination. The solid line indicates the results obtained for children without BCG scars, (---_) indicates the values for those already with BCG scars, and (- - - - - -) shows the results obtained in the children we vaccinated.

Table II. Results of the pre-vaccination (1982) and post-vaccination tests for the children vaccinated in Ahmednagar. Pre-school

Tuberculin Mean +ve size Vaccin Leprosin A School

c6 y

82

83

84

l/34 (2.9 %) 2mm o/34 10 %) -

12732 (37.5 %) 6.4+6 mm 9/32 (28.1 %) -

18/23 178.3 %) 9.6k3.2 mm 4/23 (17.4 %)

children 6-10

82 Tuberculin Mean +ve size Vaccin Leprosin A Scrofulin Flavescin Rhodesin Chitin Diernhoferin Ranin 2 Aviumin A Aviumin B Aviumin C

O/l31 0 O/27 1169 0769 l/69 O/27 O/27 o/35 o/35 1135

(1983 and 1984) skin

(0 %) (0 %.)

11-16

83

84

82

937124 (75.0 %I 6.3k3.0 mm 8727 (29.6 %I -

o/48 0 1714 o/19 o/19 l/l9 o/14 l/14 0115 o/15 o/15

7/62 4762 28762 IO/27 II27

(11.3%) (6.5 %) (45.2 %) (37.0 %) (3.7 %)

39/52 (75.0 %) 10.6k3.8 mm 17/52 (32.7 %) 25/52 (48.0 %) 15/52 (28.8 %) -

21/35

(6O:O %)

-

(1.4 %) (0 %) (1.4 %) (0 %) (0 %) (0 %) to %) (2.9 %)

y

83

84

(7.1 %)

37147 (78.8 %) 6.5k3.4 mm 804 (57.1 %) -

to %I (0%) (5.3 %) to %I (7.1 %) (0 %) (0 %) (0 %)

3718 2118 7118 7114 5714 4715 3715 1405

17120 (85.0 %) 10.7k3.1 mm 9720 (45.0 %) 1 l/20 (55.0 %) 6120 (30.0 %) -

(0 %)

(16.7 (11.1 (38.9 (50.0 (35.7 (26.7 (20.0 (93.3

%) %) %I %) %) 96) %) %)

174

Stanford

and

others

were 3.1 t-1.3 mm among the pre-school The BCG scar sizes 1 year after vaccination children, 5.9*-2.1 mm among the 6-10 year olds, and 7.Ok2.4 mm for those over 11 years. There were no visible scars in 5/196 of the vaccinated children, yet three of the five converted to Tuberculin positivity. Discussion By the criteria used, the results obtained appear to be very promising. All three age groups of children studied showed responses to Tuberculin 2 years after vaccination indistinguishable from those observed 10 years after BCG in the U.K. The children in Ahmednagar show a considerable enhancement of Tuberculin positivity after BCG vaccination. This was seen both in those already vaccinated in the preliminary survey and in those vaccinated by us. The conversion to Tuberculin positivity and the mean size of the responses achieved by the second follow up after BCG vaccination in Ahmednagar are very similar to those found in the much younger children in Agra (mean age 1.2 years) as well as to those found in adults after BCG vaccination in the UK. The sizes of responses to Tuberculin of all three vaccinated groups in Ahmednagar were significantly smaller than those found in the children with a mean age of 4.1 years in Agra [5]. The first year follow up results for the school children in Ahmednagar are very similar to those obtained in the Lebanon [91 in percentage positivity, but smaller in reaction size. By the second year the Ahmednagar results are significantly greater than those from the Lebanon. These comparisons and the statistics related to them are shown in Table III. In the first follow up, fewer of the pre-school children were Tuberculin positive than was

Table III. Table showing between the age group after BCG. Number year Ahmednagar Preschool (3.4 v) 6-10 (8.4 v) Ilf (12.8 VI Agra Preschool (1.2 v) Preschool (4.1 y) Lebanon J-17 y United Adults

the statistical significance of differences in Tuberculin responses 1 and 2 years vaccinated in different places and in data obtained

and

Mean positive

% positivity

1

year

year 2

12/32 (37.5 %) p
18123

(78.3 %)

&52 ns 1J/20

(75.0 %) (85.0 %)

“S

20/25 ns 31/37 ns

(80.0 %) ns

20/27

(74.0 %)

(84.0 %) ns

:;I36 (89.0 %) p
97/135

(72.0 %) pCO.02

47/81 (58.0 %) year 10

reaction

sizes year 2

1

6.413.6 ns 6.3k3.8 ns 6.5f3.4 ns

mm mm

p
mm

6.4f4.3 mm p
p
J.Jf2.3

p-Co.005

mm

pco.02

9.6+3.2 mm ns 10.6k3.8 mm ns 10.7+3.1 mm ns 10.424.6 mm PCO.005 14.9k6.1 mm p<0.0001 6.4k2.2 year IO

Kingdom

*Fisher’s exact test. tstudent’s t test.

214/266

(80.5 %)

9.9k4.02

BCG in ‘Ahmednagar

175

expected, and the reason for this is not clear. As can be seen from the Tables I-III, among both age groups of school children the percentage Tuberculin positivity at the second follow up is greater than in those found to have BCG scars during the initial survey (p<.O2), although there are no significant differences in mean reaction sizes. There are several possible explanations for this. Since we do not know when those already vaccinated received their BCG, the reduction in positivity could be a change with time. The difference could be due to different BCG vaccines, Madras and Glaxo, or it could be due to scars of other vaccines being confused with those of BCG resulting in non-vaccinated children being included with the vaccinated. Despite this, it is difficult not to reach the conclusion that BCG is likely to be effective in Ahmednagar, all results obtained being similar to those from the UK. A difference for which we can offer no explanation is in the sizes of BCG scars. These increase from 3.lk1.3 mm in the pre-school children to 5.9k2.1 mm (p
Table IV. A comparison between Agra, Ahmednagar and Lebanon.

the skin test results for children

Ahmednagar

Tuberculin Aviumin A Aviumin 13 Aviumin C Scrofulin Kansasin Marinin Gordonin Vaccin Leprosin A Flavescin Rhodesin Chitin Ranin 2 Diernhoferin Gilvin Nonchromogenicin -

= not tested.

Agra

Y

6-10

11r

4

2.9 %

40.5 23.9 5.8 40.8 22.1 16.2 24.3 3.6 20.3 28.6 21.6 2.7 51.4 23.5 11.8

27.2

0 % -

17.5 13.9 7.1 29.8 9.9 19.6 15.7 4.6 10.5 10.0 4.3 1.1 4.3 8.3 4.2

-

-

4

without

16.8 -

Y

BCG scars in

Lebanon 7-17

5-10

lli

65.1 65.7 22.0 93.5 73.6 52.9 68.6 36.4 42.9 30.8 -

81.6 75.3 19.5 94.8 84.1 60.3 89.0 34.5 46.6 47.6 -

4.2 8.3 2.9 8.3 5.2

-

-

1.5 1.5

176

Stanford

and

others

(p
of living the same, would seem likely

BCG the same to be an effect

Acknowledgements We should like to thank the children, their parents and teachers for allowing our studies to take place. The Evangeline Booth Hospital was the base for our studies and are deeply grateful to the director Major Y. J. Balid and his staff for their kindness and help. We should also like to thank Lepra and the MRC for supporting this project with a series of student grants. Dr R. J. W. Rees of the National Institute for Medical Research, London, generously supplied the Leprosin A. References

6 7

8

9 10

Tuberculosis Prevention Trial, Madras. (1979). Trial of BCG vaccines in South India for tuberculosis prevention. Indian Journal of Medical Research, 70, 349. Anonymous. (1980). BCG: Bad news from India. Lancer, (leader) 1, 73. Tripathy, S. P. (1986). The Chingleput BCG Trial-a 15 year report, XXWth World Conference of the lnrernarional Union against Tuberculosis, Singapore. Stanford, J. L. (19831. The BCG trials. The Practitioner, 227, 1. Stanford, J. L., Mehrotra, M. L., Cunningham, F., Pilkington, A., Sargeant, I., Series, H., Bhatti, H., Bennett, E. (1987). A prospective study of BCG (Glaxo) given to young children in Agra, India, a region of high contact with environmental mycobacteria. Tubercle, 68, 39. Eshetu, L., Stanford, J. L. (1984). Skin test sensitisation by tubercle bacilli and by other mycobacteria in Ethiopian school children. Tubercle, 66, 285. Shield, M. J., Stanford, J. L., Paul, R. C., Carswell, J. W. (1977). Multiple skin-testing of tuberculosis patients with a range of New Tuberculins, and a comparison with leprosy and Mycobacterium ulcerans infection. Journal of Hygiene, Cambridge, 78, 331. McIntyre, G., Belsey, E., Stanford, J. L. (1986). Taxonomic differences between Mycobacterium avium and Mycobacterium intracellulare elucidated in man by skin tests with three new tuberculins. furopean Journal of Respiratory Diseases, 69, 146. Bahr, G. M., Stanford, J. L., Rook, G. A. W., Rees, R. J. W., Frayha, G. J., Abdelnoor, A. M. (1986). Two potential improvements to BCG and their effect on skin test reactivity in the Lebanon. Tubercle, 67, 205. Stanford, J. L., Eshetu, L. (1983). The use of a sonicate preparation of Mycobacterium tuberculosis (New Tuberculin) in the assessment of BCG vaccination. Tubercle, 64, 275.

11 Bahr, G. M., Stanford, to mycobacteria 67. 197.

J. L., Rook, G. A. W., Rees, R. J. W., Frayha, G. J., Abdelnoor, A. M. (1986). Skin sensitisation amongst school children prior to a study of BCG vaccination in North Lebanon. Tubercle,