Background Bacille Calmette-Guérin (BCG) vaccine, primarily used against tuberculosis, has off-target (non-specific) immunomodulatory effects, including potential protection against recurrences of latent herpes simplex virus (HSV) infections. Recurrent episodes of HSV herpes labialis (oral ‘cold sores’) affect a substantial proportion of the population, including young children, and there is currently no effective preventive intervention. Methods This pilot study, nested within the MIS BAIR randomised controlled trial (RCT), explored whether neonatal BCG vaccination, compared with no BCG vaccination, reduces the incidence and recurrence of herpes labialis during the first five years of life. Findings Among the 563 children (44% of 1272 randomised) who completed the 5-year follow-up, children in the BCG group were less likely to develop herpes labialis, compared with the control group (5% vs 8%; 38% relative risk reduction, p = 0.1). Risk of recurrence was significantly lower in the BCG group (33% vs. 71%; 53% relative risk reduction, p = 0.02). BCG-vaccinated children also reported a later onset of symptoms and a reduced impact on fluid intake during episodes. Interpretation This exploratory study suggests a protective role for neonatal BCG against HSV in children. However, its limitations, including its sample size, incomplete follow up and lack of virological confirmation, mean further studies are needed to confirm these findings. Funding National Health and Medical Research Council (NHMRC) of Australia and others, as detailed in the acknowledgements.

BACKGROUND:Asthma has a significant impact worldwide, but prevention strategies remain limited. We aimed to evaluate the efficacy of neonatal BCG vaccination in preventing asthma by modulating early-life immunity. METHODS:The Melbourne Infant Study: BCG for Allergy and Infection Reduction (MIS BAIR) was a phase 3 multicentre randomized controlled trial in Victoria, Australia. Infants were randomly assigned to receive the BCG-Denmark vaccine or no intervention within 10 days of birth. The incidence of asthma at 5 years of age was estimated using the International Study of Asthma and Allergies in Childhood questions. CLINICALTRIAL:gov (NCT01906853). RESULTS:A total of 1272 infants were randomized. The adjusted incidence of asthma was 14.4% in the BCG group compared to 16.0% in the control group (adjusted risk difference [aRD] -1.7 percentage points; 95%CI -7.4, 3.9). Secondary outcomes, including severe asthma and use of preventer medication, showed similar trends, with an aRD of -3.9 (95%CI -7.7, 0.0), and -5.6 (95%CI -10.9, -0.4), respectively, favoring the BCG group. Among participants with one or both parents asthmatic, the rate of asthma was also lower in the BCG group (17.6%) compared with the control group (24.7%; aRD -7.2; 95%CI -15.9, 1.5), although a test for interaction was not significant (p = .07). CONCLUSIONS:While the point estimates suggested BCG vaccination might protect against asthma, the wide uncertainty around the estimates means further studies with larger sample sizes are needed to evaluate the long-term benefits of BCG vaccination beyond its primary indication.

BACKGROUND:Respiratory tract infections (RTI) are among the leading causes of hospitalisation and death for children under 5 years. We aimed to determine if neonatal BCG vaccination reduces early life hospitalisations for lower RTI (LRTI) in a low-tuberculosis endemic setting. METHODS:Neonates were randomised 1:1 to receive BCG-Denmark vaccination (BCG group) or no intervention (Control) in a phase 3 randomised controlled trial in Australia (NCT01906853). Hospitalisation for infection was determined by parent-reported questionnaires. The effect of BCG vaccination on the incidence of hospitalisation for LRTI in the first five years was analysed by intention-to-treat with multiple imputation. RESULTS:Complete case data to five years was available for 711 (56 %) of the 1272 participants. In the time-to-event analysis of the 1272 infants randomised, 14.2 % had one or more hospitalisations due to infection by five years. The predominant cause of hospitalisation for infection was LRTI in the first year of life (37 % of first hospitalisations), and upper RTI (33 %) by five years. In the multiple imputation analysis of the 1272 participants, the adjusted incidence of hospitalisation for LRTI in the first five years was 9.6 % in the BCG group compared to 12.4 % in the Control group (adjusted risk difference [aRD] -2.8 percentage points; 95 % CI -7.8 to 2.3). Secondary outcomes of hospitalisation for any infection and any respiratory illness were also lower in the BCG group. No interaction was observed between the effect of BCG vaccination on hospitalisation for LRTI and maternal BCG vaccination, sex or delivery mode. CONCLUSIONS:In a low-mortality setting, there was not clear evidence that neonatal BCG vaccination significantly reduces the incidence of early life hospitalisation for LRTI, infections or respiratory illness. The ability to detect any effect of BCG may have been limited by the increasing missing data over the course of follow-up.

Pneumococcal Conjugate Vaccines (PCVs) have substantially reduced the burden of disease caused by Streptococcus pneumoniae (the pneumococcus). However, protection is limited to vaccine serotypes, and when administered to children who are colonized with pneumococci at the time of vaccination, immune responses to the vaccine are blunted. Here, we investigate the potential of a killed whole cell pneumococcal vaccine (WCV) to reduce existing pneumococcal carriage and mucosal disease when given therapeutically to infant mice colonized with pneumococci. We show that a single dose of WCV reduced pneumococcal carriage density in an antibody-dependent manner. Therapeutic vaccination induced robust immune responses to pneumococcal surface antigens CbpA, PspA (family 1) and PiaA. In a co-infection model of otitis media, a single dose of WCV reduced pneumococcal middle ear infection. Lastly, in a two-dose model, therapeutic administration of WCV reduced nasal shedding of pneumococci. Taken together, our data demonstrate that WCV administered in colonized mice reduced pneumococcal density in the nasopharynx and the middle ear, and decreased shedding. WCVs would be beneficial in low and middle-income settings where pneumococcal carriage in children is high.

ABSTRACTBackgroundThe beneficial off‐target effects of Bacille Calmette–Guérin (BCG) vaccination potentially include protection against allergy.ObjectiveIn the MIS BAIR trial, we aimed to determine whether neonatal BCG vaccination reduces atopic sensitisation and clinical food allergy in infants.MethodsIn this randomised controlled trial, 1272 neonates were allocated to BCG‐Denmark vaccine (0.05 mL intradermal dose) or no BCG at birth. Randomisation was stratified by recruitment site, mode of delivery and plurality of birth. The primary outcome was the incidence of atopic sensitisation determined by skin prick test at 1 year of age. Food allergy was determined by 3‐monthly online questionnaires and oral food challenges. Data were analysed by intention‐to‐treat using binary regression. Clinicaltrials.gov (NCT01906853).ResultsAtopic sensitisation during the first year of life was 22.9% among infants in the BCG group and 18.9% in the control group (adjusted risk difference (aRD) 3.8% (95% CI −1.5 to 9.1) after multiple imputation). Clinical food allergy was similar between infants in the BCG and control groups (9.8% vs. 9.6%; aRD 0.2, 95% CI −3.4 to 3.8). An interaction was observed between the primary outcome and maternal history of BCG vaccination. No interaction was observed for the additional prespecified potential effect modifiers tested (sex, delivery mode, family history of any allergy, season of birth, hepatitis B vaccination at randomisation, BCG scar and age at BCG administration).Conclusions and Clinical RelevanceNeonatal BCG‐Denmark vaccination does not protect against atopic sensitisation or clinical food allergy in the first year of life.

Background:The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000). Methods:This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch. Results:Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal 'sentinel' surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. Conclusions:The consortium's aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies. Funding:No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210]).

Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhoeal disease in human infants. EPEC strains are defined by the presence of specific virulence factors including intimin (encoded by the eae gene) and bundle forming pili (Bfp). Bfp is encoded by the bfp operon and includes the bfpA gene for the major pilus subunit. By definition, Bfp are only present in typical EPEC (tEPEC), for which, humans are considered to be the only known natural host. This study detected tEPEC in faecal samples from a wild Australian fruit bat species, the grey-headed flying-fox (Pteropus poliocephalus). Whole genome sequencing of 61 E. coli isolates from flying-foxes revealed that 21.3 % (95%CI: 13 %-33 %) were tEPEC. Phylogenetic analyses showed flying-fox tEPEC shared evolutionary lineages with human EPEC, but were predominantly novel sequence types (9 of 13) and typically harboured novel bfpA variants (11 of 13). HEp-2 cell adhesion assays showed adherence to human-derived epithelial cells by all 13 flying-fox tEPEC, indicating that they all carried functional Bfp. Using an EPEC-specific duplex PCR, it was determined that tEPEC comprised 17.4 % (95%CI: 13 %-22 %) of 270 flying-fox E. coli isolates. Furthermore, a tEPEC-specific multiplex PCR detected the eae and bfpA virulence genes in 18.0 % (95%CI: 8.0 %-33.7 %) of 506 flying-fox faecal DNA samples, with occurrences ranging from 1.3 % to 87.0 % across five geographic areas sampled over a four-year period. The identification of six novel tEPEC sequence types and five novel bfpA variants suggests flying-foxes carry bat-specific tEPEC lineages. However, their close relationship with human EPEC and functional Bfp, indicates that flying-fox tEPEC have zoonotic potential and that dissemination of flying-fox tEPEC into urban environments may pose a public health risk. The consistent detection of tEPEC in flying-foxes over extensive geographical and temporal scales indicates that both wild grey-headed flying-foxes and humans should be regarded as natural tEPEC hosts.

Approximately 90% of chronic typhoid carriers with persistent Salmonella enterica serovar Typhi (S. Typhi) gallbladder infection have gallstones. In Samoa, where typhoid fever has been endemic for many decades, risk factors predisposing to the development of gallstones are increasing among adults. The Samoa Typhoid Fever Control Program dispatches a "Typhoid Epidemiologic SWAT Team" to perform a household investigation of every blood culture-confirmed case of acute typhoid fever. Investigations include screening household contacts to detect chronic carriers. Following limited training, two nonexpert ultrasound operators performed point-of-care ultrasound (POCUS) on 120 Samoan adults from August to September 2019 to explore the feasibility of POCUS to detect individuals with gallstones during household investigations and community screenings. POCUS scans from 120 Samoan adults in three cohorts (28 food handlers, two typhoid cases and their 18 household contacts, and 72 attendees at an ambulatory clinic) were reviewed by a board-certified radiologist who deemed 96/120 scans (80%) to be interpretable. Compared with the radiologist (gold standard), the nonexpert operators successfully detected 6/7 Samoans with gallstones (85.7% sensitivity) and correctly identified 85/89 without gallstones (95.5% specificity). The proportion (24/120) of uninterpretable scans from this pilot that used minimally trained clinicians (who are neither radiologists nor ultrasound technicians) indicates the need for additional training of POCUS operators. Nevertheless, this pilot feasibility study engenders optimism that in the Samoan setting nonexperts can be trained to use POCUS to diagnose cholelithiasis, thereby helping (along with stool cultures and Vi serology) to identify possible chronic S. Typhi carriers.

Salmonella enterica serovar Typhi (S. Typhi) is either widely distributed or proximally transmitted via fecally-contaminated food or water to cause typhoid fever. In Samoa, where endemic typhoid fever has persisted over decades despite water quality and sanitation improvements, the local patterns of S. Typhi circulation remain unclear. From April 2018-June 2020, epidemiologic data and GPS coordinates were collected during household investigations of 260 acute cases of typhoid fever, and 27 asymptomatic shedders of S. Typhi were detected among household contacts. Spatial and temporal distributions of cases were examined using Average Nearest Neighbor and space-time hotspot analyses. In rural regions, infections occurred in sporadic, focal clusters contrasting with persistent, less clustered cases in the Apia Urban Area. Restrictions to population movement during nationwide lockdowns in 2019-2020 were associated with marked reductions of cases. Phylogenetic analyses of isolates with whole genome sequences (n = 186) revealed one dominant genotype 3.5.4 (n = 181/186) that contains three Samoa-exclusive sub-lineages: 3.5.4.1, 3.5.4.2, and 3.5.4.3. Variables of patient sex, age, and geographic region were examined by phylogenetic groupings, and significant differences (p<0.05) associated genetically-similar isolates in urban areas with working ages (20-49 year olds), and in rural areas with age groups typically at home (<5, 50+). Isolates from asymptomatic shedders were among all three sub-lineages. Whole genome sequencing provided evidence of bacterial genetic similarity, which corroborated 10/12 putative epidemiologic linkages among cases and asymptomatic shedders, as well as 3/3 repeat positives (presumed relapses), with a median of one single nucleotide polymorphism difference. These findings highlight various patterns of typhoid transmission in Samoa that differ between urban and rural regions as well as genomic subtypes. Asymptomatic shedders, detectable only through household investigations, are likely an important reservoir and mobile agent of infection. This study advances a "Samoan S. Typhi framework" that supports current and future typhoid surveillance and control efforts in Samoa.

BACKGROUND:There is no standardized definition for infant eczema, and various tools have been used across studies, precluding direct comparison.OBJECTIVE:The aim of the study was to assess and to compare the accuracy of diagnostic tools for infant eczema using the extensive data collected in Melbourne Infant Study: BCG for Allergy and Infection Reduction (MIS BAIR), an eczema prevention trial.METHODS:Eczema incidence was assessed by 3 questionnaire-based measures: modified UK diagnostic tool, parent-reported medically diagnosed eczema, and parent-reported use of topical corticosteroids. Agreement between the definitions was quantified using κ coefficient. Eczema severity was assessed by 3-monthly Patient-Oriented Eczema Measure (POEM) scores and a SCORing Atopic Dermatitis (SCORAD) clinical assessment at a 12-month visit (ClinicalTrial.gov: NCT01906853).RESULTS:Among the 538 participants fulfilling at least 1 of the 3 questionnaire-based eczema definitions, only 197 participants (37%) met all 3 definitions. Agreement between the definitions was poor with κ coefficients ranging from -0.11 to 0.62. The most frequently reported symptoms were generally dry skin (483/538, 90%) and pruritus (400/538, 74%). The face (352/538, 65%) and the trunk (306/538, 57%) were more frequently affected than the creases (257/538, 48%). Participants fulfilling all 3 questionnaire-based definitions of eczema were more likely to have higher severity scores and earlier onset of symptoms.CONCLUSIONS:There is poor agreement between currently available tools for assessing infant eczema.

Introduction: Bacille Calmette-Guerin (BCG) and hepatitis B (HBV) vaccines are frequently given concomitantly at birth. Neonatal BCG vaccination induces off-target immunological effects. Whether HBV vaccine has immunomodulatory effects is unknown. As off-target effects might vary when vaccines are given simultaneously, this randomised controlled trial aimed to evaluate the influence of neonatal vaccination with BCG and/or HBV on heterologous immune responses. Methods: A total of 185 neonates in Australia were randomised to receive either neonatal BCG-Denmark vaccine, HBV vaccine, both (BCG + HBV group), or none (No vaccine group). In-vitro responses to heterologous stimulants were assessed 7 days after vaccination. The influence of (i) randomisation group and (ii) sex on interferon-gamma (IFN-c), monocyte chemoattractant protein-1 (MCP-1), and tumour necrosis factor-alpha (TNF-a) responses was analysed using linear regression. Results: Overall, BCG vaccination alone or with HBV co-administration reduced IFN-c and MCP-1 responses to heterologous stimulants. HBV vaccination alone did not alter heterologous cytokine responses. In general, males produced more IFN-c and TNF-a than females. We observed a sexdifferential effect in relation to the influence of HBV co-administration on the effect of BCG on heterologous responses. Compared with males in the No vaccine group, males in the BCG + HBV group had lower IFN-c and MCP-1 responses. In contrast, compared with females in the No vaccine group, females in the BCG group had higher IFN-c response and lower MCP-1 responses. Conclusion: Neonatal BCG vaccination resulted in lower cytokine responses to unrelated pathogens. HBV co-administration did not have a significant impact on responses overall but influenced the heterologous effects of neonatal BCG vaccination in a sex-differential manner.

Rationale: Scar formation following bacillus Calmette-Guerin (BCG) vaccination has been associated with lower all-cause mortality; the relation between scar and mycobacteria-specific protection against tuberculosis is debated. Objectives: To evaluate the association between BCG skin reaction and mycobacteria-specific immune responses. Methods: A post hoc analysis was done among 214 infants in Australia randomized to vaccination with one of three BCG vaccine strains (BCG-Denmark, BCG-Japan, or BCG-Russia) given at birth or BCG-Denmark given at 2 months of age. Measurements and Main Results: BCG skin reaction size and characteristics 10 weeks after vaccination were related to the in vitro mycobacteria-specific immune responses measured in stimulated whole blood. The size and characteristics of the skin reaction correlated positively with in vitro immune responses, even after adjusting for BCG vaccine strain and age at vaccination. Specifically, the reaction size and characteristics correlated with the proportion of mycobacteria-specific polyfunctional CD41 T cells after stimulation with BCG and PPD and, to a lesser extent, after stimulation with Mycobacterium tuberculosis or Mycobacterium ulcerans. A similar correlation was observed with concentrations of IFN-gamma, IL-2, tumor necrosis factor, and IL-13 in the supernatant after stimulation with BCG, PPD, and M. tuberculosis and to some degree for the proportions of mycobacteria-specific polyfunctional CD8(+) T cells and CD107(+) cytotoxic cells. Conclusions: BCG skin reaction correlated with the magnitude of mycobacteria-specific T-cell responses. As T-cell responses play a key role in defense against mycobacteria, the relationship between BCG scar formation and protection against tuberculosis should be revisited. This may also extend to the need for BCG revaccination in scar-negative individuals.

In this study, we used whole genome sequencing and comparative genomics analyses to characterize the population structure, evolutionary origins, and genomic features of S. Typhi associated with decades of endemic typhoid fever in Samoa.

Abstract Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhoeal disease in human infants. EPEC is characterised by specific virulence factors including intimin (encoded by the eae gene) and bundle forming pili (Bfp; encoded by the bfp operon, including the bfpA gene for the major pilus subunit). By definition, Bfp are only present in typical EPEC (tEPEC). Humans are the only known natural host of tEPEC. Here we investigated the occurrence and genetic characteristics of EPEC from an Australian fruit bat species, the grey-headed flying-fox. Whole genome sequencing of 61 E. coli isolates from fruit bat faeces revealed that 13 were tEPEC, 11 of which had novel bfpA variants. HEp-2 cell adhesion assays indicated that every tEPEC strain carried a functional bfp operon that encoded localised adherence to human-derived epithelial cells in vitro. Additional analysis of faecal samples using an EPEC-specific multiplex PCR, identified tEPEC in fruit bats from three geographic areas in eastern Australia, with sampling conducted on six occasions over a four-year period (mean occurrence 19.1%, range 1.3–87.0%). This is the first report of the widespread occurrence of tEPEC in a non-human host over extensive geographical and temporal scales. Our findings suggest that fruit bats are a natural host of tEPEC and that host-specific lineages may have evolved in bats to comprise part of their commensal microbiome.

Bacille Calmette‐Guérin (BCG) vaccine could play a role in counteracting the rising prevalence of atopic diseases, through its beneficial off‐target effects. We aimed to determine whether neonatal BCG vaccination reduces the incidence of eczema in infants.

Enterotoxigenic E. coli (ETEC) is a common cause of diarrhea in children in low- and middle-income countries, and in travelers to these countries. ETEC is also an important cause of morbidity and premature mortality in piglets, calves, goat kids and lambs. The major virulence determinants of ETEC are enterotoxins and colonization factors, which enable the pathogen to colonize the small intestine and deliver enterotoxins, such as the heat-stable enterotoxins, STp and STh, to epithelial cells. Because most ETEC strains are host-specific, there are few convenient animal models to investigate the pathogenesis of ETEC infections or to evaluate specific anti-ETEC interventions, such as drugs and vaccines. An exception is ETEC strains bearing F41 pili, which mediate intestinal colonization of various young animals, including neonatal mice, to cause disease and in some cases death. In this study, we used the archetypal F41-producing bovine ETEC strain, B41 (O101:NM; K99, F41, STp) to validate and further explore the contribution of F41 and STp to bacterial virulence. By using targeted gene deletion and trans-complementation studies, augmented by whole genome sequencing, and in vitro and animal studies of virulence, we established that F41 mediates colonization of the mouse intestine and is essential for bacterial virulence. In addition, we showed for the first time that STp is as important as F41 for virulence. Together, these findings validate the use of neonatal mice to study the pathogenesis of F41-bearing ETEC and to investigate possible specific anti-ETEC interventions including vaccines that target heat-stable enterotoxins.

Background. Bacille Calmette-Guerin (BCG) vaccination has beneficial off-target effects that may include protecting against non-mycobacterial infectious diseases. We aimed to determine whether neonatal BCG vaccination reduces lower respiratory tract infections (LATI) in infants in the Melbourne Infant Study: BCG for Allergy and Infection Reduction (MIS BAIR) trial. Methods. In this investigator-blinded trial, neonates in Australia were randomized to receive BCG-Denmark vaccination or no BCG at birth. Episodes of LRTI were determined by symptoms reported in parent-completed, 3-month questionnaires over the first year of life. Data were analyzed by intention-to-treat using binary regression. Results. A total of 1272 neonates were randomized to the BCG vaccination (n = 637) or control (n = 635) group. The proportion of participants with an episode of LRTI in the first year of life among BCG-vaccinated infants was 54.8% compared to 58.0% in the control group, resulting in a risk difference of -3.2 (95% confidence interval, -9.0 to 2.6) after multiple imputation. There was no interaction observed between the primary outcome and sex, maternal BCG, or the other prespecified effect modifiers. Conclusions. Based on the findings of this trial, there is insufficient evidence to support the use of neonatal BCG vaccination to prevent LRTI in the first year of life in high-income settings.