Heliyon 9 (2023) e15241 Contents lists available at ScienceDirect Heliyon journal homepage: www.cell.com/heliyon Research article Factors influencing scar formation following Bacille Calmette-Guérin (BCG) vaccination Paola Villanueva a, b, c, d, *, Nigel W. Crawford a, b, d, e, Mariana Garcia Croda f, Simone Collopy g, Bruno Araújo Jardim h, Tyane de Almeida Pinto Jardim h, Laurens Manning i, j, k, Michaela Lucas j, l, m, n, Helen Marshall o, Cristina Prat-Aymerich p, q, Alice Sawka r, Ketaki Sharma s, t, Darren Troeman p, Ushma Wadia i, Adilia Warris u, v, Nicholas Wood s, t, w, Nicole L. Messina a, b, 1, Nigel Curtis a, b, c, 1, **, Laure F. Pittet a, b, c, x, 1 a Department of Paediatrics, The University of Melbourne, Parkville, VIC, Australia b Infection and Immunity, Murdoch Children’s Research Institute, Parkville, VIC, Australia c Infectious Diseases, Royal Children’s Hospital Melbourne, Parkville, VIC, Australia d Department of General Medicine, Royal Children’s Hospital Melbourne, Parkville, VIC, Australia e Immunisation Service, Royal Children’s Hospital Melbourne, Parkville, VIC, Australia f School of Medicine, Federal University of Mato Grosso do Sul, Campo Grande, MS, Brazil g Department of Pediatrics, Universidade Estadual do Rio de Janeiro, Rio de Janeiro, Brazil h Carlos Borborema Clinical Research Institute, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil i Wesfarmers Centre for Vaccines and Infectious Diseases, Telethon Kids Institute, Perth, WA, Australia j School of Medicine, University of Western Australia, Perth, WA, Australia k Department of Infectious Diseases, Fiona Stanley Hospital, Perth, WA, Australia l Department of Immunology, Sir Charles Gairdner Hospital, Perth, WA, Australia m Departments of Immunology, Perth Children’s Hospital, Perth, WA, Australia n Department of Immunology, Pathwest, QE2 Medical Centre, Perth, WA, Australia o Robinson Research Institute and Adelaide Medical School, The University of Adelaide and Department of Paediatrics, The Women’s and Children’s Health Network, Australia p Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands q Institut d’Investigació Germans Trias i Pujol, Departament de Genètica i Microbiologia, CIBER de enfermedades respiratorias (CIBERES), Instituto de Salud Carlos III, Universitat Autònoma de Barcelona, Catalunya, Badalona, Spain r Department of Thoracic Medicine, Royal Adelaide Hospital, Adelaide, SA, Australia s National Centre for Immunisation Research and Surveillance, Westmead, NSW, Australia t The Children’s Hospital at Westmead, Westmead, NSW, Australia u Medical Research Council Centre for Medical Mycology, University of Exeter, UK v Great Ormond Street Hospital, London, UK w The Children’s Hospital at Westmead Clinical School, Faculty of Medicine and Health, University of Sydney, NSW, Australia x Infectious Diseases Unit, Department of Paediatrics, Gynaecology and Obstetrics, Faculty of Medicine, University of Geneva and University Hospitals of Geneva, Geneva, Switzerland * Corresponding author. ** Corresponding author. Department of Paediatrics, The University of Melbourne, Parkville, VIC, Australia. E-mail addresses: paola.villanueva@rch.org.au (P. Villanueva), nigel.curtis@rch.org.au (N. Curtis). 1 Joint senior authors. https://doi.org/10.1016/j.heliyon.2023.e15241 Received 18 October 2022; Received in revised form 29 March 2023; Accepted 30 March 2023 Available online 6 April 2023 2405-8440/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). P. Villanueva et al. Heliyon 9 (2023) e15241 A R T I C L E I N F O A B S T R A C T Keywords: The prevalence of scar formation following Bacille Calmette-Guérin (BCG) vaccination varies BCG vaccine globally. The beneficial off-target effects of BCG are proposed to be stronger amongst children BCG scar who develop a BCG scar. Within an international randomised trial (‘BCG vaccination to reduce Vaccination technique the impact of coronavirus disease 2019 (COVID-19) in healthcare workers’; BRACE Trial), this Vaccine safety nested prospective cohort study assessed the prevalence of and factors influencing scar formation, as well as participant perception of BCG scarring 12 months following vaccination . Amongst 3071 BCG-recipients, 2341 (76%) developed a BCG scar. Scar prevalence was lowest in Spain and highest in UK. Absence of post-injection wheal (OR 0.4, 95%CI 0.2–0.9), BCG revaccination (OR 1.7, 95%CI 1.3–2.0), female sex (OR 2.0, 95%CI 1.7–2.4), older age (OR 0.4, 95%CI 0.4–0.5) and study country (Brazil OR 1.6, 95%CI 1.3–2.0) influenced BCG scar prevalence. Of the 2341 participants with a BCG scar, 1806 (77%) did not mind having the scar. Participants more likely to not mind were those in Brazil, males and those with a prior BCG vaccination history. The majority (96%) did not regret having the vaccine. Both vaccination-related (amenable to optimisation) and individual-related factors affected BCG scar prevalence 12 months following BCG vaccination of adults, with implications for maximising the effectiveness of BCG vaccination. 1. Introduction Bacille Calmette-Guérin (BCG) vaccine is widely administered in over 150 countries to protect children against tuberculosis (TB) [1]. A small characteristic scar at the BCG injection site, which develops over several weeks to months, is considered a normal response and commonly used as a surrogate for effective vaccination [2]. The importance of scar formation is highlighted by studies that link the protective ‘off-target’ (also known as ‘non-specific’) clinical effects of BCG vaccination to the development of a scar [3,4]. In observational studies in low-income countries, BCG-vaccinated children who developed a scar had lower all-cause mortality and fewer hospital admissions than those who did not [5–9]. In addi­ tion, the presence and size of BCG scar have been shown to correlate with the magnitude of the immune response to BCG vaccination [10] . The prevalence of scar following BCG vaccination varies and the mechanisms underlying this are unclear. Suggested explanations include variation in immune response, the influence of BCG strain and administration technique [9,11–16]. The need for revaccination in scar-negative children is debated [2,13,17]. With increasing interest in BCG vaccination and revaccination for broader uses in both children and adults, it is important to understand more about BCG scarring. Within an international randomised controlled trial of BCG vaccination to reduce the impact of coronavirus disease 2019 (COVID- 19) in healthcare workers (the BRACE trial; ClinicalTrials.gov NCT04327206), this nested prospective cohort study aimed to deter­ mine (a) the prevalence of BCG scarring at 12 months following vaccination, (b) the factors influencing scar formation, and (c) participant perception of scarring. 2. Methods 2.1. Setting and participants The BRACE trial recruited healthcare workers (HCW) in Australia, Brazil, Spain, the Netherlands and the United Kingdom (UK) from March 2020 to April 2021, and randomised participants to receive BCG vaccine or no BCG. HCW were eligible if working in healthcare settings or having face-to-face contact with patients during the COVID-19 pandemic. Exclusion criteria comprised any contra-indication to BCG, including immunosuppression or previous significant local BCG adverse reaction. Prior BCG vaccination, or previous history of positive tuberculin skin test (TST), were not exclusion criteria. The trial protocol is described in detail elsewhere [18]. 2.2. Intervention Participants randomised to BCG received a single dose of BCG-Denmark (AJ Vaccines, Copenhagen), 0.1 ml (corresponding to 2–8 × 105 colony-forming units of Mycobacterium bovis, Danish strain 1331) intradermally in the upper arm, using a short (10 mm) bevel needle (25 G to 30 G). If an individual had prior BCG scar evidence at recruitment, the vaccinators were instructed to administer the vaccine or placebo a minimum of 2.5 cm from the original BCG scar. All vaccinators were trained in intradermal delivery of BCG vaccine (Supplemental Material 1). If a post-injection wheal (minimum 7 mm diameter) [19] did not occur immediately, a participant could receive a second vaccine dose. Participants were informed about the normal expected injection site reaction, including likely scar formation. Participants recruited in Australia from March to May 2020 were also required to receive a single intramuscular dose (0.5 ml, pre-filled syringe) of a quadrivalent inactivated influenza vaccine in the contralateral upper arm on the day of randomisation. 2 P. Villanueva et al. Heliyon 9 (2023) e15241 2.3. Data collection Data were collected using Research Electronic Data Capture (REDCap) web application [20] including details on demographics, co-morbidities, previous BCG vaccination, previous TST and previous known latent tuberculosis infection (LTBI). Data on the presence of post-injection wheal formation (including injection site photograph), BCG batch, and number of participants BCG vaccinated per vaccinator, were collected. Information on prior BCG vaccination experience (before the BRACE trial) was collected from vaccinators in Brazil and Spain. Information on injection site scar formation (presence and scar descriptors), vaccine site photographs (with ruler or standard coin for scale; Fig. 1(A-G)) and participant scar perception were solicited from participants using a standardised web-based questionnaire 12 months following vaccination (Supplemental Material 2). Participants who reported an ‘abnormal thick scar’ had injection site photos reviewed by a medical doctor, to assess for keloid scar formation. 2.4. Case definitions The following definitions were used. BCG-revaccination: BCG vaccination in a participant who had any prior BCG vaccination history. Post-injection wheal: a skin wheal of minimum 7 mm diameter immediately following intradermal vaccination [19]. Imperfect BCG vaccine administration: the absence of a post-injection site wheal. Keloid scar: a thick raised scar extending upwards and outwards well beyond the site of vaccination. 2.5. Statistical analysis StataIC 14.0 (Statacorp LP, College Station, TX, USA) was used. BCG scar prevalence at 12 months following BCG vaccination was calculated among participants who provided injection site data. BCG scar prevalence was evaluated by study country and by individual vaccinator (defined as proportion of vaccinees with scar presence at 12 months). To identify factors (participant-related and vaccination-related) associated with BCG scar formation, odds ratio (OR) and 95% confidence intervals (CI) were determined using univariate logistic regression. Post-injection wheal presence, as a potential associated factor, was analysed amongst participants who received one BCG dose only. Significant factors (p-value <0.2) resulting from the univariate logistic regression analysis were included as possible covariates in a multivariate logistic regression model. The model was created using backward stepwise exclusion of factors with p-value >0.05, using sequential model testing. Participant BCG scar descriptors and scar perception were analysed amongst participants with a BCG scar at 12 months following BCG vaccination. Ethical approval was obtained from The Royal Children’s Hospital Human Research Ethics Committee (HREC 62586) with sub­ sequent approvals from all participating sites. All participants provided signed informed consent prior to enrolment. 3. Results Of the 3411 participants who were BCG-vaccinated in the BRACE trial, 3071 (90%) provided injection site information in their 12- month questionnaire (Fig. 2 (A and B)). They ranged in age from 18 to 78 years (median 41) and the majority (76%) were female (Table 1). Figure 1. BCG scar description at 12 months. Representative photographs for BCG scar descriptions. (A) skin colour mark without redness (normal scar formation), (B) red mark, (C) red mark with crusting, (D) red mark with discharge, (E) purple mark, (F) inflamed appearance with surrounding swelling and/or redness, (G) abnormal thick scar. 3 P. Villanueva et al. 4 Figure 2. BRACE participants who received BCG in A) Stage 1 and B) Stage 2. Abbreviations: BCG, Bacille Calmette-Guérin; BRACE trial, BCG vaccination to reduce the impact of coronavirus disease 2019 (COVID-19) in healthcare workers; dTpa, diphtheria-tetanus-acellular pertussis vaccine, reduced antigen formulation; 12MQ, 12-month questionnaire. Heliyon 9 (2023) e15241 P. Villanueva et al. Heliyon 9 (2023) e15241 Table 1 Demographics and factors investigated for association with BCG scar formation. Factor Total BCG Scar prevalence Univariate Multivariate (N=3071) n/N (%) OR (95% CI) OR (95% CI) Sex Male 822 556 (67.6) 1 (reference) 1 (reference) Female 2249 1785 (79.4) 1.84 (1.53–2.20), p < 0.001 2.00 (1.65–2.42), p < 0.001 Age 18–49 2144 1733 (80.8) 1 (reference) 1 (reference) ≥50 927 608 (65.6) 0.45 (0.38–0.54), p < 0.001 0.43 (0.35–0.51), p < 0.001 Nutritional status (BMI) Normal weight (18.5–24.9) 1266 964 (76.1) 1 (reference) – Underweight (<18.5) 35 28 (80.0) 1.25 (0.54–2.90), p = 0.6 Pre-obesity (25.0–29.9) 1090 820 (75.2) 0.95 (0.79–1.15), p = 0.6 Obesity class I (30.0–34.9) 419 328 (78.2) 1.13 (0.87–1.47), p = 0.4 Obesity class II (35.0–39.9) 155 121 (78.1) 1.11 (0.75–1.67), p = 0.6 Obesity class III (≥40) 58 45 (77.6) 1.08 (0.58–2.04), p = 0.4 Unknown 48 35 (72.9) NA Smoker No 2808 2131 (75.9) 1 (reference) – Yes 263 210 (79.8) 1.26 (0.92–1.72), p = 0.2 Diabetes No 2991 2278 (76.2) 1 (reference) – Yes 80 63 (78.8) 1.16 (0.67–1.99), p = 0.6 Chronic respiratory disease No 2866 2177 (76.0) 1 (reference) – Yes 205 164 (80.0) 1.27 (0.89–1.80), p = 0.2 Chronic cardiovascular disease No 2740 2107 (76.9) 1 (reference) – Yes 331 234 (70.7) 0.72 (0.56–0.93), p = 0.01 Study country Australia 1380 1003 (72.7) 0.70 (0.59–0.83), p < 0.001 0.84 (0.70–1.01), p= 0.07 Brazil 1222 1032 (84.4) 2.24 (1.86–2.69), p < 0.001 1.61 (1.29–2.01), p< 0.001 Netherlands 280 187 (66.8) 0.59 (0.46–0.77), p < 0.001 1.01 (0.75–1.36), p= 0.9 Spain 110 52 (47.3) 0.26 (0.18–0.39), p < 0.001 0.31 (0.20–0.46), p< 0.001 UK 79 67 (84.8) 1.76 (0.95–3.28), p = 0.07 1.84 (0.97–3.50), p= 0.06 BCG history 1st BCG 990 677 (68.4) 1 (reference) 1 (reference) BCG revaccination 2081 1664 (80.0) 1.85 (1.55–2.19), p < 0.001 1.65 (1.33–2.04), p < 0.001 Previous known LTBI No 3031 2309 (76.2) 1 (reference) – Yes 23 16 (69.6) 0.71 (0.29–1.74), p = 0.5 Unknown 17 16 (94.1) NA Previous TST Negative/None 2568 1961 (76.4) 1 (reference) – Positive (>5 mm) 186 149 (80.1) 1.25 (0.86–1.81), p = 0.2 Unknown 317 231 (72.9) NA BCG batch 118006D 591 431 (72.9) 0.80 (0.66–0.99), p = 0.04 – 118017F 820 587 (71.6) 0.71 (0.60–0.86), p < 0.001 118019D 658 536 (81.5) 1.48 (1.19–1.84), p = 0.001 119039B 82 70 (85.4) 1.84 (0.99–3.42), p = 0.06 119053A 631 527 (83.5) 1.75 (1.39–2.20), p < 0.001 200731–014 245 160 (65.3) 0.56 (0.42–0.73), p < 0.001 200904–017 35 27 (77.1) 1.05 (0.48–2.33), p = 0.9 Unknown 9 3 (33.3) NA Co-administered influenza vaccine† No 1883 1490 (79.1) 1 (reference) – Yes 1188 851 (71.6) 0.67 (0.57–0.79), p < 0.001 Post-injection wheal* Yes 2898 2223 (76.7) 1 (reference) 1 (reference) No 32 19 (59.4) 0.44 (0.22–0.90), p = 0.03 0.44 (0.21–0.93), p = 0.03 Unknown 141 99 (70.2) NA NA Vaccinator experience ≥20 vaccinees 2608 2007 (77.0) 1 (reference) – 0-19 vaccinees 463 334 (72.1) 0.76 (0.61–0.95), p = 0.02 Abbreviations: BCG, Bacille Calmette-Guérin; BMI, body mass index; OR, odds ratio; LTBI, latent tuberculosis infection; NA, not applicable; TST, tuberculin skin test. *Wheal response (yes/no) analysed for participants who received one BCG dose only. †Stage 1 participants (Australia) were required to receive influenza vaccination on day of randomisation. Significant factors (p-value <0.2) resulting from the univariate logistic regression analysis were included as possible covariates in a multivariate logistic regression model. The model presented in the table was created using backward stepwise exclusion of factors with p-value >0.05, using sequential model testing. 5 P. Villanueva et al. Heliyon 9 (2023) e15241 3.1. BCG scar prevalence Overall, BCG scar prevalence at 12 months was 76% (2341/3071); lowest (47%, 52/110) in Spain and highest (85%, 67/79) in the UK (Fig. 3). Most scars were visible to participants (as opposed to only palpable) (96%, 2254/2341; Table 2) and were reported as a ‘skin colour mark without redness’ by 71% (1651/2341) of participants (Table 3; Fig. 1(A)). Scars were reported as ‘abnormally thick’ by 3% (60/2341). Of these 60 participants, 22 supplied an injection site photo that showed hypertrophic scarring. Two of these (one each in Australia and Brazil) had a keloid scar at their BCG injection site, one of whom had prior predisposition to keloid scarring. 3.1.1. Scar prevalence according to vaccinator Amongst the total of 114 vaccinators, those whose vaccinees had a scar prevalence greater than 50% at 12 months, were more prevalent in Brazil (36/38, 95% of vaccinators) and least prevalent in Spain (2/8, 25% of vaccinators) (Fig. 4). In Brazil, 7/38 (18%) vaccinators had prior experience of working in BCG clinics, in addition to specific vaccination training for the BRACE trial. They vaccinated 33% of participants in Brazil. In Spain, none of the 8 vaccinators reported prior experience of working in BCG clinics. 3.2. Factors associated with the development of BCG scar In the univariate analysis, BCG scar formation at 12 months was more common amongst female participants, those with a history of prior BCG vaccination, participants in Brazil, and those vaccinated with certain BCG batches (Table 1). BCG scar formation was less likely in older participants, those with chronic cardiovascular disease, those who had influenza vaccine co-administered, those with imperfect BCG vaccine administration (absence of post-injection wheal), those vaccinated by vaccinators who administered fewer BCG vaccines in the trial, and those vaccinated in Australia, Netherlands or Spain. In the multivariate analysis, older age group (OR 0.43, 95% CI 0.35–0.51), female sex (OR 2.00, 95%CI 1.65–2.42), study country (Brazil OR 1.61, 95% CI 1.29–2.01; Spain OR 0.31, 95% 0.20–0.46), BCG revaccination (OR 1.65, 95% CI 1.33–2.04), and absence of post-injection wheal (OR 0.44, 95% CI 0.21–0.93) influenced BCG scar prevalence (Table 1). There was no significant association in either analysis with participant nutritional status, smoking, certain co-morbidities, previous positive TST or LTBI (Table 1). Sensitivity analyses using participants only in Australia (supplemental Table S1) and those only in Brazil (supplemental Table S2) (the countries with the largest number of participants) also showed, in multivariate analyses, that sex, age group and BCG revacci­ nation influenced BCG scar prevalence. Figure 3. BCG scar prevalence at 12 months by recruitment country, with 95% confidence interval. Table 2 BCG scar visibility and palpability at 12 months, as reported by participants. Scar visibility and palpability n = 2341 Visible only, not palpable 1267 (54%) Visible and palpable 970 (41%) Palpable only, not visible 74 (3%) Visible and palpable with crust 17 (<1%) Unknown 13 (<1%) 6 P. Villanueva et al. Heliyon 9 (2023) e15241 Table 3 BCG scar description at 12 months Scar description n = 2341 Skin colour mark without redness (normal scar formation) 1651 (71%) Red mark 602 (26%) Red mark with crusting 14 (<1%) Red mark with discharge 3 (<1%) Purple mark 7 (<1%) Inflamed appearance with surrounding swelling and/or redness 4 (<1%) Abnormal thick scar 60 (3%) Ulcer 0 (0%) Total Scar prevalence Factor BCG Univariate Multivariate n = 1380 n/N (%) OR (95% CI) OR (95% CI) Sex Male 353 219 (62.0) 1 (reference) 1 (reference) Female 1027 784 (76.3) 1.97 (1.52–2.56), p < 0.001 2.04 (1.54–2.71), p < 0.001 Age 18–49 912 701 (76.9) 1 (reference) 1 (reference) ≥50 468 302 (64.5) 0.55 (0.43–0.70), p < 0.001 0.41 (0.31–0.54), p < 0.001 Nutritional status (BMI) Normal weight (18.5–24.9) 621 471 (75.8) 1 (reference) – Underweight (<18.5) 11 7 (63.6) 0.56 (0.16–1.93), p= 0.4 Pre-obesity (25.0–29.9) 451 311 (69.0) 0.71 (0.54–0.93), p= 0.01 Obesity class I (30.0–34.9) 163 114 (69.9) 0.74 (0.51–1.09), p= 0.1 Obesity class II (35.0–39.9) 62 47 (75.8) 1.00 (0.54–1.84), p= 0.9 Obesity class III (>40) 32 26 (81.3) 1.38 (0.56–3.42), p= 0.5 Unknown 40 27 (67.5) NA Smoker No 1298 948 (73.0) 1 (reference) – Yes 82 55 (67.1) 0.75 (0.47–1.21), p = 0.2 Diabetes No 1359 988 (72.7) 1 (reference) – Yes 21 15 (71.4) 0.94 (0.36–2.44), p = 0.9 Chronic respiratory disease No 1276 920 (72.1) 1 (reference) – Yes 104 83 (79.8) 1.53 (0.93–2.51), p = 0.1 Chronic cardiovascular disease No 1274 934 (73.3) 1 (reference) – Yes 106 69 (65.1) 0.68 (0.45–1.03), p = 0.07 BCG history 1st BCG 651 463 (71.1) 1 (reference) 1 (reference) BCG revaccination 729 540 (74.1) 1.16 (0.92–1.47), p = 0.2 1.58 (1.19–2.08), p = 0.001 Previous known LTBI No 1353 983 (72.7) 1 (reference) – Yes 15 9 (60.0) 0.56 (0.20–1.60), p = 0.3 Unknown 12 11 (91.7) NA Previous TST Negative/None 1021 739 (72.4) 1 (reference) – Positive (>5 mm) 115 88 (76.5) 1.24 (0.79–1.96), p = 0.3 Unknown 244 176 (72.1) NA BCG batch 118006D 591 431 (72.9) 1.02 (0.80–1.30), p = 0.9 – 118017F 789 572 (72.5) 0.98 (0.77–1.24), p = 0.9 Co-administered influenza vaccine† No 192 152 (79.2) 1 (reference) 1 (reference) Yes 1188 851 (71.6) 0.66 (0.46–0.96), p = 0.03 0.57 (0.39–0.84), p < 0.01 Post-injection wheal* Yes 1231 899 (73.0) 1 (reference) 1 (reference) No 19 11 (57.9) 0.51 (0.20–1.27), p = 0.1 0.34 (0.13–0.90), p = 0.03 Unknown 130 93 (71.5) NA – Vaccinator experience ≥20 vaccinees 1199 864 (72.1) 1 (reference) – 0-19 vaccinees 181 139 (76.8) 1.28 (0.89–1.85), p = 0.2 Total Scar prevalence Factor BCG Univariate Multivariate n = 1222 n/N (%) OR (95% CI) OR (95% CI) Sex Male 350 279 (79.7) 1 (reference) 1 (reference) (continued on next page) 7 P. Villanueva et al. Heliyon 9 (2023) e15241 Table 3 (continued ) Total Scar prevalence Female 872 753 (86.3) 1.61 (1.16–2.23), p < 0.01 1.65 (1.18–2.30), p < 0.01 Age 18–49 977 856 (87.6) 1 (reference) 1 (reference) ≥50 245 176 (71.8) 0.36 (0.26–0.51), p < 0.001 0.37 (0.26–0.52), p < 0.001 Nutritional status (BMI) Normal weight (18.5–24.9) 387 327 (84.4) 1 (reference) – Underweight (<18.5) 18 15 (83.3) 0.59 (0.26–3.27), p = 0.9 Pre-obesity (25.0–29.9) 498 418 (83.9) 0.96 (0.67–1.38), p = 0.8 Obesity class I (30.0–34.9) 212 187 (88.2) 1.37 (0.83–2.26), p = 0.2 Obesity class II (35.0–39.9) 81 66 (81.5) 0.81 (0.43–1.51), p = 0.5 Obesity class III (>40) 21 14 (66.7) 0.37 (0.14–0.95), p = 0.04 Unknown 5 5 (100.0) NA Smoker No 1090 916 (84.0) 1 (reference) – Yes 132 116 (87.9) 1.38 (0.80–2.38), p = 0.3 Diabetes No 1167 988 (84.7) 1 (reference) – Yes 55 44 (80.0) 0.72 (0.37–1.43), p = 0.4 Chronic respiratory disease No 1162 980 (84.3) 1 (reference) – Yes 60 52 (86.7) 1.21 (0.56–2.58), p = 0.6 Chronic cardiovascular disease No 1047 894 (85.4) 1 (reference) – Yes 175 138 (78.9) 0.64 (0.43–0.95), p = 0.03 BCG history 1st BCG 40 31 (77.5) 1 (reference) – BCG revaccination 1182 1001 (84.7) 1.61 (0.75–3.43), p = 0.2 Previous known LTBI No 1218 1028 (84.4) 1 (reference) – Yes 1 1 (100.0) NA† Unknown 3 3 (100.0) NA Previous TST Negative/None 1150 970 (84.3) 1 (reference) – Positive (>5 mm) 42 38 (90.5) 1.76 (0.62–5.00), p = 0.3 Unknown 30 24 (80.0) NA BCG batch 118019D 658 536 (81.5) 0.60 (0.44–0.83), p < 0.01 0.63 (0.45–0.87), p < 0.01 119039B 3 3 (100.0) NA† NA† 119053 A 557 491 (88.2) 1.71 (1.23–2.35), p = 0.001 1.58 (1.13–2.19), p < 0.01 Unknown 4 2 (50.0) NA NA Post-injection wheal* Yes 1212 1024 (84.4) 1 (reference) No 6 6 (100.0) NA† Unknown 4 2 (50.0) NA Vaccinator experience ≥20 vaccinees 1098 926 (84.3) 1 (reference) – 0–19 vaccinees 124 106 (85.5) 1.09 (0.65–1.85), p = 0.7 3.3. Participant scar perception Amongst participants with a BCG scar at 12 months, 1806 (77%) ‘[did] not mind having the scar at all’, 472 (20%) would ‘rather not have a scar but [understood] this [was] unavoidable’, and 59 (3%) were ‘dissatisfied with the scar’ (Supplemental Material 3). Brazil had the highest proportion of participants (97%) who ‘[did] not mind having the scar at all’ (Fig. 5). Those BCG revaccinated were more likely to accept scarring, compared with those receiving it for the first time (1382/1661 (83%) vs 424/676 (63%), p < 0.001). Males were more likely to ‘not mind having the scar at all’, compared with females (475/553 (89%) vs 1331/1784 (75%), p < 0.001). Of the 59 participants who were ‘dissatisfied with the scar’, 46 (78%) reported this was because the scar was ‘worse than [I] expected’, 10 (17%) ‘did not expect to have a scar’, and 3 (5%) reported other reasons. Other reasons were related to scar location, odd and different appearance (purple/red scar colour) to that expected. The majority of participants with a scar at 12 months (96%, 2242/ 2341) ‘[did] not regret having the vaccine [because of the scar]’. 4. Discussion In this large international study, we found that both vaccination-related and individual-related factors influence scar formation following BCG vaccination. Studies of scar prevalence following BCG vaccination of adults are scarce and limited by small numbers of vaccinees. In our study of 8 P. Villanueva et al. Heliyon 9 (2023) e15241 Figure 4. Scar prevalence by vaccinator, grouped by study country. Proportion of scar positive vaccinees at 12 months, per vaccinator. Each data point represents an individual vaccinator. Horizontal lines represent medians. Figure 5. BCG scar perception at 12 months by study country. more than 3000 vaccinated healthcare workers, 76% of BCG-Denmark-vaccinated participants reported the presence of a scar at 12 months, with significant differences by country. This is lower than the scar prevalence of 99% reported amongst 175 healthcare students who were BCG-vaccinated 12 months prior with BCG-Denmark in Sweden [21]. Notably, the students were younger (mean age 24 years old) compared with those in our study (mean 42 years old). In children, reported scar prevalence varies from 99% [11] (BCG-Denmark) to as low as 52% [8] (BCG-Russia). Studies have implicated mainly vaccine-related factors such as BCG strain, vaccination route and dose [10,11,15,22–27]. BCG strains, derived from the original M. bovis BCG strain first used in 1921, have acquired phenotypic and genotypic variations during decades of in vitro culturing under diverse conditions in laboratories around the world [28,29]. Studies have shown different immunological responses and different mycobacterial viability according to strain [30–33]. We found that age, sex, study country, prior history of BCG vaccination and presence of post-injection wheal may all influence the development of a BCG scar. Vaccination technique and vaccinator experience have previously been reported to affect the local BCG injection site reaction in children [9,11,12,15,25]. Intradermal BCG vaccination is a difficult technique to master and the presence of a post-injection wheal is a marker of intradermal delivery [19]. Consistent with studies in children, we found the absence of a wheal was associated with a decreased likelihood of scar formation, highlighting the importance of a correct vaccine administration. We found an association between number of vaccines given by vaccinators and the likelihood of BCG scar formation, although this was no longer significant in the multivariate analysis. However, different levels of background experience existed amongst vaccinators, in addition to the training received specifically for the trial. For example, in Brazil, the only study country with an ongoing universal neonatal BCG immunisation program, a third of trial participants were vaccinated by vaccinators who had additional prior experience of working in BCG clinics. Consistent with this hypothesis, more vaccinators in Brazil than any other study country had a scar prevalence of >50% amongst their participants, whereas Spain (with the lowest BCG scar prevalence) had the least proportion of such vaccinators. The higher likelihood of BCG scarring in participants with prior BCG vaccination history may relate to an underlying 9 P. Villanueva et al. Heliyon 9 (2023) e15241 immunological boosting phenomenon, as BCG revaccination has been associated with more pronounced local injection site reactions [34], larger scar size [35] and enhanced protective effects (against respiratory tract infections) in studies in adolescents [36] and adults [37]. BCG injection site reactions (presence and size) have been shown to correlate with the magnitude of the mycobacteria-specific T-cell immune responses [10] as well as specific and heterologous cytokine responses [9] in vitro. The decreased likelihood of BCG scarring amongst older participants and males may also relate to differing immune responses according to age and sex [38,39]. For older participants, immunosenescence may play a role [40]. Our finding accords with another study comparing individuals BCG-vaccinated at greater than 60 years old with younger adults in Malawi [35]. Sex-differential BCG scar prevalence has been described in two studies in children, showing lower scar formation amongst girls compared with boys [8,41], but not in four other studies [15,21,25,35]. Sex-related differences in beneficial effects of BCG vaccination have also been reported amongst infants; some studies showing the beneficial effects favour the male sex (randomised controlled trials in Uganda [42] and Guinea-Bissau [43]) and others the female sex [6,7,44], Nonetheless, in interpreting our finding of decreased likelihood of scarring amongst males compared with females, we acknowledge the possibility of gender-related differences in reporting behaviour in our study, as men have been shown to underreport on health-related matters and thus may be less likely to notice or report a small scar on their arm [45]. Moreover, the majority of participants in our study were female. Scar perception differed by country, prior BCG scar experience and sex. Brazil, a high TB prevalence country, had the highest proportion of participants who accepted scarring. This may be due to the active national infant BCG vaccination program normalising BCG scars in the population. There may also be differences in cosmetic scar appearance according to ethnic skin type [46,47]. Males were also less concerned with their BCG scar, consistent with a smaller study assessing scar acceptance amongst high-school-aged children in the UK [41]. This study has some limitations. Scar size was not assessed, and this has previously been shown to correlate with the extent of the underlying immune response [10] and the beneficial off target (non-specific) effects in infants [9,48]. Scar prevalence at 12 months was assessed by participants, which may affect accuracy of scar detection (the self-reporting nature may explain the observed sex-difference), although they were HCW previously informed of the expected injection site reaction at recruitment and were asked to provide injection site photographs. Furthermore, potential confounding factors could include vaccinators’ prior BCG vaccination experience, potential variances in vaccine administration technique, as well as BCG batch. The strengths of this study include the prospective data collection of vaccine site reactions in a large number of individuals across multiple countries, vaccinated with the same BCG strain. Our findings have implications for BCG vaccination campaigns as well as the growing number of trials into the beneficial off-target effects of BCG in both adults and children [49–53]. Optimising vaccine-related factors, particularly correct intradermal administration leading to a wheal, can increase the likelihood of scar development and consequent protective effects of BCG vaccination. 5. Conclusion BCG scar prevalence following BCG vaccination in adults was affected by several vaccination-related (vaccine technique, prior BCG, study site) and individual-related (sex, age at vaccination) factors. Although participant BCG scar perception varied by country, sex and prior BCG vaccination, the vast majority of participants did not regret having the vaccine. Author contribution statement Conceptualisation or design of the work: Paola Villanueva, Laure F. Pittet, Nicole L. Messina and Nigel Curtis. Acquisition of data: all authors. Analysis or interpretation of data: Paola Villanueva and Laure F. Pittet. Original drafting: Paola Villanueva. Revising, editing, and final approval of the manuscript: all authors. Data availability statement Data included in article/supp. material/referenced in article. Acknowledgements We thank the BRACE trial participants for making this study possible. We also thank the researchers involved in establishing the BRACE trial (see Supplemental Material 4 for the BRACE trial commenced in Australia), in particular: Ms. Veronica Abruzzo, Ms. Sonja Elia, Mr. Richard Hall, Ms. Casey Goodall, Dr. Ellie McDonald, Ms. Ann Krastev, Dr. Samantha Bannister, Ms. Grace Gell, Dr. Wendy Norton, Dr. Joyce Chan, A/Prof. Peter Richmond, Prof. Tobias Kollmann, Dr. Susan Hermann, Ms. Erin Latkovic, Ms. Michelle England, Dr. Roberto Oliveira, Dr. Estela Carvalho, Dr. Helen Catterick, Ms. Glauce Dos Santos, Ms. Catriona Doran, Dr. Alison Gifford, Dr. Telma Goldenberg, Dr. Christina Guo, Dr. Georgina Newman, Dr. Ligia Olivio, Dr. Lorrie Symons, Dr. Niki Tan, Dr. Laura Tate, Dr. Tina Zhou. The Murdoch Children’s Research Institute (MCRI) leads the BRACE trial across five countries. It is supported by the Victorian Government’s Operational Infrastructure Support Programme. The trial is also supported by the Bill & Melinda Gates Foundation [INV- 017302], the Minderoo Foundation [COV-001], Sarah and Lachlan Murdoch, the Royal Children’s Hospital Foundation [2020–1263 BRACE Trial], Health Services Union NSW, the Peter Sowerby Foundation, the Ministry of Health Government of South Australia is in Australia, the NAB Foundation, the Calvert-Jones Foundation is in Australia, the Modara Pines Charitable Foundation, the UHG Foundation Pty Ltd, Epworth Healthcare and individual donors. The sponsors had no role in the collection, analysis and interpretation 10 P. Villanueva et al. Heliyon 9 (2023) e15241 of data or in the preparation, review or approval of the manuscript. P.V. is supported by the Australian Government Research Training Programme Scholarship provided by the Australian Commonwealth Government and the University of Melbourne, and an MCRI Ph.D. Top-Up Scholarship. L.F.P. is supported by the Swiss National Science Foundation is based in Switzerland [Early Postdoc Mobility Grant, P2GEP3_178155]. N.C. is supported by a National Health and Medical Research Council Investigator Grant [GNT1197117]. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.heliyon.2023.e15241. References [1] A. Zwerling, M.A. Behr, A. Verma, T.F. Brewer, D. Menzies, M. Pai, The BCG World Atlas: a database of global BCG vaccination policies and practices, PLoS Med. 8 (2011), e1001012. [2] J. Jason, L.K. Archibald, O.C. Nwanyanwu, P.N. Kazembe, J.A. Chatt, E. Norton, H. Dobbie, W.R. Jarvis, Clinical and immune impact of Mycobacterium bovis BCG vaccination scarring, Infect. Immun. 70 (2002) 6188–6195. [3] A. 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