R. J. W. Rees

Two potential improvements to BCG and their effect on skin test reactivity in the Lebanon

An account of an ongoing project to assess the possible benefits of two additives to BCG vaccine is presented. These additives are suspensions of irradiation killed Mycobacterium vaccae in one case, and M. leprae in the other. Groups of children aged 7-17 living in Zgharta and Akkar districts of North Lebanon have received vaccination with BCG alone or with either of the two additives since 1980. This region was chosen since contact with environmental mycobacteria is small, but both leprosy and tuberculosis occur there. So far the effects of the additives have been assessed by annual skin testing of volunteers with Tuberculin, Leprosin A, Vaccin and Scrofulin, and by measuring the size of the vaccine scars. Some children have now been followed up on four occasions, and special attention is paid to them. No complications have been encountered in the 1740 children who have entered the study (by our observation, or by local report on those who have not attended for follow-up) and the mean scar sizes after vaccines with the additives are no larger than those after BCG alone. There is no evidence that the additives have prevented development of Tuberculin positivity after vaccination, or have changed the nature of reaction to it. Incorporation of M. leprae significantly increased Leprosin A positivity and both additives increased Vaccin positivity in comparison with the effects of BCG alone. The results are fitted to a model of the theoretical expectations of the study and may be beginning to show the advantages expected of the additives. The only unexpected finding was a reduction in Scrofulin positivity especially associated with the additives. The very low contact with environmental mycobacteria experienced in the study area has allowed the pattern of post-vaccination decay of skin test positivity to be studied with greater precision than has been reported before, and differences have been detected between the two districts where the study was conducted. Confirmation of the possible advantage of the additives will rest with data to be obtained in longer term follow-ups and in studies being carried out in other countries.

Skin sensitization to mycobacteria amongst school children prior to a study of BCG vaccination in North Lebanon

1888 school children aged between 7 and 17 years, living in 13 villages in two districts of North Lebanon, were skin tested with four new tuberculins as the initial step in a study of BCG vaccination. The great majority of children were tested with Tuberculin, Leprosin A, Vaccin and Scrofulin. In comparison with other countries where similar studies have been carried out, extremely low levels of sensitization were discovered, indicating very little contact with mycobacteria. There was, however, a statistically significant increase in positivity with increasing age. The results obtained for the villages of each district were significantly different from each other, positivity being greatest in Akkar district for each reagent. The eight villages of Zgharta district could be separated into a lowland group of four villages, a mountain group of three villages and one anomalous mountain village. There was significantly more positivity in the lowland than in the mountain villages. In Akkar district, where leprosy has a low prevalence, positivity to Leprosin A was 8% amongst the children (leaving out an anomalous village). In Zgharta district where the disease does not occur, positivity was 3.4% to Leprosin A for the lowland villages and 0.9% for the group of 3 mountain villages. The two anomalous villages were the only ones in which tuberculosis cases were known to have occurred recently, and they were the only two villages in which Tuberculin positivity exceeded 10%.

Enhancement of experimental tuberculosis in the mouse by suramin

Summary Because of the favourable effect of suramin, a powerful trypanocidal agent, which had been reported in patients with pulmonary tuberculosis, its action on the tubercle bacillus in vitro and on experimental tuberculosis in the mouse was investigated. Suramin was found to have no growth-inhibiting or promoting action on cultures in vitro , nor to have a therapeutic effect in the mouse. On the contrary, it increased the susceptibility of mice to both heavy and light tuberculous infections and caused the breakdown of established pulmonary tuberculosis. Furthermore, BCG multiplied more freely in the tissues of mice receiving suramin than in untreated animals. The possible mode of action of suramin in experimental tuberculosis is discussed, and it is suggested that it may alter the lipid surface of the tubercle bacilli in vivo so that they are less susceptible to destruction by the host defence system. This hypothesis is strengthened by the finding that suramin modifies the surface lipids of red blood cells.

The inhibition of growth of avirulent strains of Mycobacterium tuberculosis by a surface-active polyoxyethylene ether.

SUMMARY: Examination of the action of a series of surface-active polyoxyethylene ethers on the growth of the avirulent strain of Mycobacterium tuberculosis, H37 Ra, showed that one member, the ether of 2,2′-dihydroxy-5, 5′-di-tert-octyldiphenyl-methane (D4), inhibited this strain but not the virulent variant H37 Rv. This type of differential inhibition has been observed with a commercial surface-active mixture, ‘Triton A20’ (Dubos & Middlebrook, 1948). Lengthening of the polyoxyethylene chains of D4 led to loss of its inhibitory property. Both D4 and ‘Triton A20’ could induce resistance in H37 Ra, and cross-resistance to D4 of a strain resistant to ‘Triton A20’ was observed; this cross-resistance suggested that a compound closely related to D4 was the active constituent of ‘Triton A20′. The resistant strains grew in a microscopically amorphous pattern, like their parent strain. The growth of four other avirulent strains of tubercle bacilli was inhibited, like that of H37 Ra, by D4, while two slightly virulent strains grew freely in this detergent, like H37 Rv. One factor common to the strains naturally insusceptible to D4, but absent from those inhibited, is the ability to produce some degree of microscopic ‘cord’ pattern, and this property may have some connexion with the cause of the differential inhibition by D4. Surface activity also probably plays a part.

The virulence of South Indian tubercle bacilli in mice and guinea-pigs infected by the intravenous route

Summary Cultures of tubercle bacilli from 7 South Indian patients, selected for their low virulence by the intramuscular route in the guinea-pig, and from 6 British patients, were tested for their virulence in the mouse by injecting 0·1 mg. and 0·025 mg. intravenously. With the higher dose no differences were found between the Indian and British cultures in the proportions of mice that died from tuberculosis or in their median survival periods. With the lower dose there was a suggestion that the mice infected with Indian cultures survived longer. Guinea-pigs were infected intravenously with cultures from 2 Indian and 2 British patients, and the number of viable organisms in their spleens were counted at intervals thereafter. Following an initial 2 weeks of growth in the spleens, during which there were only small differences in the growth rates of virulent and attenuated bacilli, the counts on the most attenuated of the Indian cultures decreased, whereas the remaining 3 cultures continued to multiply and killed the guinea-pigs. The failure to demonstrate the difference in virulence between cultures from Indian and British patients in the mouse is attributed mainly to the route of infection and a consequent lower dosage in the visceral organs. The manifestations in experimental tuberculosis of attenuation shown by isoniazid-sensitive cultures from Indian patients and by isoniazid-resistant cultures from patients of other races are closely similar.

Changes in Morphology of an Avirulent Strain of Mycobacterium tuberculosis under the Influence of Non-Ionic Surface-Active Agents.

SUMMARY: When the avirulent strain of Mycobacterium tuberculosis H37 Ra was repeatedly subcultured in a liquid medium containing low concentrations of ‘Triton A20’ or other chemically similar non-ionic surface-active polyoxyethylene ethers, the morphology changed from the amorphous growth pattern characteristic of avirulent tubercle bacilli to the formation of microscopic ‘cords’ similar to those shown by virulent strains such as H37Rv. Once developed, the cord-forming property of H37 Ra persisted when cultures were transferred to liquid medium devoid of surface-active agent, even when such transfer was made after passage through mice; after further subcultures, however, a tendency to reversion was apparent. No difference in virulence was detected between cord-forming H37 Ra and the unmodified strain when tested in mice. Previous work had indicated that a lower member of the polyoxyethylene ether series, the dimer ‘D4’, inhibited amorphous-growing avirulent tubercle bacilli, including H37 Ra, but not the growth of naturally cord-forming strains, exemplified by the virulent H37 Rv and attenuated strains derived from it. The growth of cord-forming avirulent organisms, developed from H37 Ra in the present experiments, was also found to be relatively unaffected by D4.