The conventional methods of assessment of post-vaccination allergy by doing tuberculin testing among the vaccinated group are inapplicable in case of BCG vaccination without prior tuberculin test (Direct BCG). Because of obvious technical and operational advantages of direct BCG vaccination a search for a method of technical assessment of BCG vaccination is important. Hence, a study was carried out by the BCG Assessment Team of National Tuberculosis Institute in Tumkur district of Mysore state where Mass BCG Campaign was going on. Four groups of persons aged 0-20 years, each group belonging to two BCG Technicians area and vaccinated one day prior to visit of assessment team, were randomly selected. Besides, persons (0-20 years) from 2 unvaccinated villages of adjacent area were included as control groups. All persons were registered simultaneously tuberculin tested with 1 TU RT 23 and 5 TU RT 22 within 24 hours of BCG vaccination (for pre-vaccination allergy) and retested with tuberculin 5 TU RT 22 at the end of 3 weeks and 3 months (for post-vaccination allergy). The four vaccine groups were vaccinated with vaccine batch Nos. 977, 978, 981 and 984 respectively. Classification of the directly vaccinated persons into previously infected and non-infected by tuberculin test administered within 24 hours of vaccination and about 12 weeks later, elicitation of post-vaccination allergy only among the non-infected, has been considered as the Reference Test for judging the suitability of other methods of assessment studied. The main findings are: (1) The Reference Test showed that the four batches of BCG vaccine used had induced varying levels of allergy. (2) Assessment based on the mean size of post-vaccination reactions among 0-4 years age group, which consists predominantly of previously non-infected persons, showed a different pattern of differences between the four batches of vaccine as compared to the Reference Test. Moreover, to get adequate number of children aged 0-4 years, it will be necessary to cover a comparatively large population. (3) The method of using the mean size of post-vaccination reactions among those classified as non-infected on the basis of vaccination reactions of size 0-13 mm at the site of BCG vaccination on the 4th day of vaccination showed results similar to the Reference Test. But this method has only a marginal operational advantage over the Reference Test. (4) Using size of induration at the site of vaccination on 21st day of vaccination did not give the same results as the Reference Test. Operationally this method would have been most suitable as it involved only one visit to the group. (5) Differences between mean size of post-vaccination tuberculin reactions among directly vaccinated persons and mean size of (natural) allergy in reactors among neighbouring unvaccinated areas showed the same results as the Reference Test. This method has the operational advantage but needs further investigations. (6) Tuberculin testing of all directly BCG vaccinated persons including the natural reactors about 12 weeks after vaccination compared favourably, with the reference method, as the tuberculin reactors contributed less than 1 mm over and above the allergy in the vaccinated non-reactors . This method would be useful when rate of tuberculin reactors is less than 20% in 0-20 years age group and their mean size is also less than 20 mm. Operationally, it is a simpler method next only to No.4 above. Further investigations are considered necessary for final selection of this or any of the other methods.

The Freeze-Dried BCG vaccine manufactured in India is sealed under vacuum. This though adds to its stability involves expensive production procedures. Sealing in presence of nitrogen is both simpler and economical. Before producing this vaccine for use on a large scale, it was considered necessary, to study the influence of storage at higher temperatures on the allergy inducing capacity on the basis of the size of local lesion and viable counts of Freeze-Dried BCG vaccine sealed either in vacuum or in the presence of nitrogen. For this, half of the ampoules of a batch of vaccine prepared in Madras BCG Vaccine Laboratory were sealed in vacuum and the other in presence of nitrogen. Randomly selected ampoules of both types of vaccine were exposed to 37o and 44o for 2, 6, and 18 weeks and another set at 4oC for 18 weeks. Two batches of liquid BCG vaccine were made as controls: 16 types of ampoules thus obtained were randomly repeated 5 times according to Standard Lattice Design. About 3000 school children without BCG scar, aged 5-14 years In Bundi and Kota districts of Rajasthan were vaccinated as per the study design. post-vaccination allergy with 5 TU RT 22 by measuring the size of vaccination lesions was recorded 3 months later. Viable counts on samples of ampoules from Freeze-Dried BCG vaccines exposed differently were done in the production laboratory after 18 weeks of storage.

The vaccine in 16 types of ampoules was significantly different. Liquid BCG vaccine resulted in higher level of allergy and larger vaccination lesions than Freeze-Dried BCG vaccine sealed under either method. The study has shown that Freeze-Dried BCG vaccine sealed under either method vacuum or nitrogen, gave satisfactory level of post-vaccination allergy and induration size of vaccination lesions, provided the vaccine was preserved at 4oC. Storage at 37o for more than 2 weeks and even 2 weeks storage at 44oC affected both types of vaccine badly as shown by post-vaccination allergy and viable counts. However, decrease in viable count with time and temperature was more pronounced in vaccine sealed in presence of nitrogen. Hence, there is a need to provide cold chain facility for Freeze-Dried vaccine all throughout the period.

One of the hypothesis put forth for the failure of BCG vaccine to show protection against bacillary pulmonary tuberculosis in Chingleput trial was the interference from non-tuberculous mycobacteria that were prevalent in the trial area. In order to test this, a study was conducted with the following objectives: to investigate (1) Protection given by BCG and M.avium intracellulare (MAI) which is the most prevalent species, against the challenge with high and low virulent strains of M.tuberculosis in sensitised guineapigs. (2) Whether M.avium Intracellulare (MAI) interferes with the protective effect of BCG against challenge with both high and low virulent strains of M.tuberculosis. Sensitization was done with MAI in guineapigs using both oral and intradermal routes. Groups of species were immunized with BCG/Placebo and later challenged with high/low virulent strains of M.tuberculosis. Colony counts of M.tuberculosis bacilli from spleens of the animals were done to measure the protective effect.
The findings were: (1) BCG showed protection against both high and low virulent challenges. (2) MAI in both oral and intradermal routes had no effect against low virulent challenge. (3) There was no significant interaction between BCG and MAI against low virulent challenge. (4) MAI when given orally, showed a significant protection against high virulent challenge. The same was not seen with intradermal route. (5) MAI orally, interfered with the protective effect of BCG against high virulent strains of M.tuberculosis.