{'fraction_non_alphanumeric': 0.030717351318209688, 'fraction_numerical': 0.026180257510729613, 'mean_word_length': 5.047830923248053, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
BCG re-vaccination in Malawi: 30-year follow-up of a large, randomised, double-blind, placebo-controlled trial

Summary Background A large, double-blind, randomised, placebo-controlled trial of repeat BCG found 49% efficacy against leprosy but no protection against tuberculosis after 6–9 years’ follow-up in 1995. We report here additional follow-up, which resulted in greatly increased tuberculosis case numbers, and allowed subgroup analysis. Methods Nearly 47 000 individuals of all ages living in northern Malawi with a BCG vaccine scar were randomly assigned (1:1) between 1986 and 1989 to receive a second BCG or placebo. The investigators and project staff remained masked to all interventions. Enhanced passive surveillance ensured ascertainment of tuberculosis and leprosy to the end of 2018. Tuberculosis case definitions included rigorous microbiological or histological confirmation. Prespecified subgroup analyses were by tuberculosis type, age at vaccination, time since vaccination, previous tuberculin reactivity, HIV status and Mycobacterium tuberculosis lineage. The original trial is registered with ISRCTN registry, ISRCTN11311670. Findings In follow-up until Dec 31, 2018, 824 participants had developed tuberculosis, including 786 with pulmonary disease, of whom 383 (63%) of 607 with known HIV status were HIV positive. There was no effect of a second BCG overall (odds ratio [OR] 0·92; 95% CI 0·80–1·05), or for pulmonary (0·93; 0·81–1·07), or lymph node tuberculosis (0·60; 0·31–1·17). The OR was lower for those with known HIV-negative tuberculosis (0·77; 0·59–1·00), for those vaccinated as children (aged <5 years, 0·74; 0·41–1·35; aged 5–14 years, 0·77; 0·60–0·99), and for cases arising at least 20 years after vaccination (0·79; 0·63–1·01). There were no differences by tuberculin status at vaccination, or lineage. There was no evidence of protection against leprosy beyond 10 years after vaccination (although there have been only nine diagnostically certain cases since 1995). Interpretation There was no evidence that repeat BCG vaccination provides appreciable protection against overall tuberculosis in this rural African population with a high prevalence of HIV. Subgroup effects should not be overinterpreted given the multiple analyses done. However, the evidence for modest protection against HIV-negative tuberculosis, and for a delayed benefit in those vaccinated as children, is consistent with other observations in the literature. Funding LEPRA, Wellcome Trust, Bill & Melinda Gates Foundation.


Further notes on background to the trial
The Karonga Prevention Trial (KPT) recruitment took place 1986-1989, having been planned in the early 1980s. The proposal was driven initially by leprosy interests, led by the Immunology of Leprosy (IMMLEP) Steering Committee of the WHO TDR Programme. Thus the trial was based within a large leprosy project, known first as the Lepra Evaluation Project and later as the Karonga Prevention Study, which had been evaluating serological and skin tests developed through the IMMLEP programme since 1979. [1][2][3][4] This project had incorporated tuberculosis case finding, and its laboratory provided sputum smear services since 1980, introducing culture in 1986. [5] There was thus a framework for a trial with tuberculosis as well as leprosy outcomes. Several studies had already shown that BCG vaccines could impart a variable degree of protection against leprosy, in different populations, and there was particular interest in the IMMLEP programme as to whether the addition of leprosy bacillus antigens might enhance this protection . [6] The project also provided an opportunity to evaluate giving a second BCG vaccination, a policy which was then practiced for tuberculosis control in several countries, but which had never been evaluated in a randomised controlled trial. The trial was designed and initiated before HIV was recognised in the trial population.
BCG vaccination had been introduced into Karonga District in the late 1970s, first in campaigns which targeted schoolchildren, and then as part of the routine childhood immunization programme organised by the EPI . [7] There was evidence from case control and cohort analyses that a single BCG vaccine imparted approximately 50 % protection against leprosy, but no protection against tuberculosis in the Karonga population . [5,8] The original design was for four groups: BCG versus BCG plus killed M leprae vaccines in individuals lacking scar evidence of prior BCG vaccination, and BCG versus placebo in individuals with scar evidence of prior BCG. Recruitment began in January 1986 with this basic design, with the combined vaccine including 5 x 10 7 killed M leprae per dose. This was changed, on recommendation of the WHO IMMLEP programme, in July 1986, when a higher dose of killed M leprae (6 x 10 8 bacilli) was introduced, first for the scar negative participants, and later (in August 1987) as an additional arm among scar positive individuals. This change was introduced after pilot investigations had shown that the higher dose was safe . [6] The M leprae suspension vaccine was prepared at the Wellcome Laboratories, Beckenham, UK. [9,10]

Surveillance and follow-up by vaccine group
Follow-up of the trial population was through a mixture of active and passive surveillance. The active surveillance component consisted of annual house-to-house surveys, carried out 1991 -1994, in four areas of the district each with 5,000 -10,000 population, known to have had a relatively high incidence of leprosy. In addition, all individuals who were either first degree relatives of, or known to have lived in the same house as, a known leprosy patient since 1980, were followed up in early 1995, wherever they then lived, and examined. Furthermore, 19,471 participants in the trial lived in the area selected for a demographic surveillance site (the Karonga Continuous Registration System), established in 2002. [11] The initial survey for this surveillance site included an effort to trace all individuals who were known to have lived in the area at the time of trial recruitment 1986-1989, and recorded dates of death and emigration.

A B C D
(Note: the up-turns at the ends of each graph are an artefact due to events (deaths or migrations) being reported and recorded in the dataset before each re-census)

Tuberculosis case definitions
Changes in laboratory methods have required a change in tuberculosis case definitions compared to those used in the 1995/6 analysis. The following changes were agreed by the Data Monitoring Committee for the 2020 analyses.

Diagnostic certainty criteria for tuberculosis
In 1995/6, the following definitions were used: Extrapulmonary: certain (C) if histology "certain" or aspirate is positive on microscopy or culture/Xpert/genotype (excluding single scanty smear); probable (P) if histology "probable" or clinical lymph node tuberculosis and the aspirate has a scanty positive smear.
Note: Pulmonary means pulmonary +/-other sites "Lymph node" means involving lymph nodes with or without other extrapulmonary sites, but without pulmonary involvement.

Criterion for postvaccination status of tuberculosis cases
In 1995/6, the following definition was used: "A case was considered as postvaccination if there was no recorded evidence of tuberculosis until at least 60 days after vaccination.". [10] In that analysis there was an interest in the possible precipitation of TB by BCG vaccination, so any early cases (between 60 days and 6 months) were important to include if we had seen them previously without evidence of TB. This required individual checks of such cases. No such increase in early cases was found associated with vaccine, so there is no need to explore this again. By removing the need to have seen people prior to vaccination we can include some cases who were previously excluded. And a 6-month cut-off for all gives more certainty that the cases are actually post-vaccination. The following definitions are thus used in the current analyses:  All cases occurring within 6 months (182 days) of vaccination are excluded.
 All cases with a history of TB prior to vaccination (or at an unknown date) by either documentation or self-report are excluded.
 Only the first episodes of certain or probable TB after vaccination are included

CONSORT flow chart
The CONSORT flow chart for the complete trial with reference to tuberculosis outcomes, for both the intention to treat (ITT) and per protocol (PP) analyses, is presented in the main paper as Figure 1.

Repeat BCG and placebo comparison
The distributions of pulmonary tuberculosis cases by age and HIV status at time of diagnosis are shown for the placebo and BCG recipients in Figure  b. BCG (382)

Intention to treat analysis
The flow chart for the intention to treat analysis is in the main paper ( Figure 1), along with the odds ratios for the various comparisons, for all 824 certain and probable tuberculosis cases ( Figure 2). The distributions by study arm for age at vaccination, sex and TST status at vaccination are shown below, in Table S

Evaluation of BCG plus killed M leprae vaccines against tuberculosis
The BCG plus killed M leprae vaccines were developed and incorporated into the trial with intention to prevent leprosy, but it is appropriate to examine the results of these vaccines also versus tuberculosis, given that they represent the addition of mycobacterial antigens to BCG, and there is evidence of heterologous protection against tuberculosis associated with environmental mycobacteria. [12] The evaluation of the combined BCG plus killed M leprae vaccine was made complicated by the change in design described in Section 1, above. Not only were there two doses of killed M leprae (5 x 10 7 and 6 x 10 8 ) but a combined vaccine arm was introduced for scar positives.
Three comparisons were thus agreed for the data analysis plan by the Data Monitoring Committee: In order to ensure these comparisons are made between groups vaccinated concurrently, the analyses were stratified by time period when the compared vaccines were used.

In scar negative and scar positive individuals combined
The odds ratio for pulmonary tuberculosis, comparing BCG plus killed M leprae to BCG, combining all data in scar negatives and scar positives is 0.95 (0.86 -1.06), indicating no overall effect (Table S-5).

Combining BCG and BCG plus killed M leprae versus placebo, in scar positives
Given no evidence that the addition of killed M leprae antigens influences the protection imparted by BCG alone against tuberculosis (Table S-5), we combine the BCG and BCG+ killed M leprae groups and compare them to placebo recipients in scar positives (Table S-6).
There is no evidence of protection overall against tuberculosis overall in this combined analysis: OR= 0.95 (0.83 -1.09). This was the same after adjustment for time period before and after 01 July 1986.

Discussion
We again encourage caution in interpretation, given the multiple comparisons involved in these analyses. Regarding the implications of adding killed M leprae antigens to BCG, the overall impression is of no consistent or significant effects in these analyses, as evident in 3.4.3 which included both scar negatives and scar positives. Trends by age and by time since vaccination appear to go in the opposite direction in the scar positive and scar negative groups, and an apparent increased risk of HIV negative tuberculosis associated with BCG+KML compared to BCG alone in those with BCG scars (Figure S-7) is not seen in those without scars (Figure S-6). We conclude that the addition of M leprae antigens to BCG vaccine had no observable effect on protection against tuberculosis. To the best of our knowledge this trial is the only published examination of the efficacy of BCG plus killed M leprae vaccines against tuberculosis.

Ascertainment and diagnosis of leprosy
The trial took place within a large leprosy project which had begun in 1979. [13] Much effort had been devoted to establishing rigorous diagnostic criteria for leprosy, leading to an algorithm for diagnostic certainty based upon clinical findings, a clinical certainty score given by a medical officer, and an independent certainty assessment by a histopathologist. [13] This algorithm was used for diagnostic confirmation for all the leprosy cases in the trial analyses.
The first trial analysis took place in late 1995, after 6 -9 years of follow up, at which time 107 post-vaccination leprosy cases had been ascertained in the entire trial population. Of these, 93 were considered diagnostically certain. [10] Compared to placebo among participants with a BCG scar, the second BCG imparted 49 % protection against diagnostically certain paucibacillary and multibacillary leprosy (35 cases, RR=0.51; 95 % CI=0.25 -1.03). Given that there was observational study evidence that a single BCG imparted at least 50 % protection against leprosy in this population, this implied that two BCG vaccinations could induce some 75% protection. The fact that both the case-control and cohort observational studies had shown that protection was stronger against diagnostically certain than against certain and probable cases combined provided the rationale to focus these analyses on diagnostically certain cases. This was stipulated explicitly in the plans for the 1995 and most recent analyses. Biopsies were no longer collected routinely and the diagnostic certainty assessment was carried out by experienced Leprosy Control Assistant staff. Given that the project was well known for leprosy work in the district since 1979, and the fact that project staff working throughout the district were experienced in leprosy diagnosis, it is likely that ascertainment remained reasonably high, but some cases may have been missed.

CONSORT flow chart
The Consort flow chart of the complete trial with reference to leprosy outcomes is presented as

BCG vs placebo among BCG scar-positives
This is discussed in main paper, with reference to the ITT population. In the per-protocol analysis, which excluded 3761 individuals who received a vaccine inconsistent with their recorded scar status, results were very similar (Figure S-9). protection similar to that provided by BCG. [12] It was noted in 1995, and in this analysis, that protection against leprosy appeared to decline with age at vaccination, an observation which is consistent with the fact that protection was greatest among those with little or no tuberculin reactivity, and the fact that cumulative exposure to environmental as well as to tubercle mycobacteria rises with age. The evidence for strong protection (100 %, though based upon only 8 cases) among individuals who were tuberculin negative at vaccination is consistent with this influence of exposure to other mycobacterial antigens.

Discussion
In addition to the Karonga trial, two other trials were organised to evaluate the efficacy of BCG plus killed M leprae vaccine against leprosy, in Venezuela, and in South India. The Venezuela trial compared the combination vaccine against BCG alone among 29,113 contacts of leprosy patients (many of whom had already received a BCG vaccination). No effect on leprosy incidence was observed in this comparison. [14] The South India trial took place in a general population, where 171,400 individuals were randomised to receive one of five interventions: placebo, BCG, BCG + killed M leprae, "ICRC", or "Mw" (the latter two were killed preparations of different environmental mycobacterial species). Efficacy of BCG alone and of the BCG + killed M leprae vaccines were reported to be 34.1 % (95% CI 13.5 -49.8), and 64 % (50.4 -73.9), respectively There was no formal comparison between BCG+ killed M leprae and BCG alone, but these results imply a relative efficacy of the combined vaccine versus BCG alone of 45 %. [15,16] Neither trial was designed to evaluate protection against tuberculosis.
Once again we see examples of differing efficacy of mycobacterial vaccines between different populations. The immunological reasons for these differences continue to be debated, amid repeated calls for the experiences with vaccines against tuberculosis and leprosy to be considered together in order to guide research for the future. [17]