Cross-sectional health centre and community-based evaluation of the impact of pneumococcal and malaria vaccination on antibiotic prescription and usage, febrile illness and antimicrobial resistance in young children in Malawi: the IVAR study protocol.

INTRODUCTION: Vaccination is a potentially critical component of efforts to arrest development and dissemination of antimicrobial resistance (AMR), though little is known about vaccination impact within low-income and middle-income countries. This study will evaluate the impact of vaccination on reducing carriage prevalence of resistant Streptococcus pneumoniae and extended spectrum beta-lactamase-producing Escherichia coli and Klebsiella species. We will leverage two large ongoing cluster-randomised vaccine evaluations in Malawi assessing; first, adding a booster dose to the 13-valent pneumococcal conjugate vaccine (PCV13) schedule, and second, introduction of the RTS,S/AS01 malaria vaccine. METHODS AND ANALYSIS: Six cross-sectional surveys will be implemented within primary healthcare centres (n=3000 users of outpatient facilities per survey) and their local communities (n=700 healthy children per survey): three surveys in Blantyre district (PCV13 component) and three surveys in Mangochi district (RTS,S/AS01 component). We will evaluate antibiotic prescription practices and AMR carriage in children ≤3 years. For the PCV13 component, surveys will be conducted 9, 18 and 33 months following a 3+0 to 2+1 schedule change. For the RTS,S/AS01 component, surveys will be conducted 32, 44 and 56 months post-RTS,S/AS01 introduction. Six health centres in each study component will be randomly selected for study inclusion. Between intervention arms, the primary outcome will be the difference in penicillin non-susceptibility prevalence among S. pneumoniae nasopharyngeal carriage isolates in healthy children. The study is powered to detect an absolute change of 13 percentage points (ie, 35% vs 22% penicillin non-susceptibility). ETHICS AND DISSEMINATION: This study has been approved by the Kamuzu University of Health Sciences (Ref: P01-21-3249), University College London (Ref: 18331/002) and University of Liverpool (Ref: 9908) Research Ethics Committees. Parental/caregiver verbal or written informed consent will be obtained prior to inclusion or recruitment in the health centre-based and community-based activities, respectively. Results will be disseminated via the Malawi Ministry of Health, WHO, peer-reviewed publications and conference presentations.

Invasiveness potential of pneumococcal serotypes in children after introduction of PCV13 in Blantyre, Malawi.

INTRODUCTION: The introduction of PCV13 to the Malawi infant immunization schedule in 2011 has been associated with reduced disease from Streptococcus pneumoniae. Improved understanding of serotypes with high invasive potential can guide future vaccination interventions. We aimed to estimate pneumococcal serotypes associated with acute respiratory infection (ARI) and invasive pneumococcal disease (IPD) in hospitalized children in Blantyre, Malawi. METHODS: We analysed data from healthy children under 5 years in the community in Blantyre and children admitted to Queen Elizabeth Central Hospital with ARI between 2015 and 2018. Nasopharyngeal swabs from children were tested for S. pneumoniae and serotyped by latex agglutination if positive. We analysed culture-positive blood and cerebrospinal fluid samples from admitted children between 2012 and 2018 to identify cases of IPD after the introduction of PCV13. We calculated the age-adjusted odds ratio (OR) of carriage for S. pneumoniae vaccine serotypes (VT) comparing those with ARI to healthy children. We also calculated age-adjusted ORs comparing serotypes causing IPD to carriage in the community with OR > 1 indicating high invasive potential. RESULTS: Serotypes 5 (OR 24.73 [95% CI 7.90-78.56] p < 0.001), 1 (OR 23.38 [95% CI 9.75-56.06] p < 0.001), and 6B (OR 4.73 [95% CI 1.66-11.64] p = 0.001) had high invasive potential. Serotype 6B was no longer significant (OR 1.34 [95% CI 0.07-6.87] p = 0.777) in a sensitivity analysis accounting for year of recruitment. The prevalence of S. pneumoniae carriage in the community was 72.6% [95% CI 71.3-74.0] (3078/4238) and 23.4% (719/3078) of positive community samples were VT. The carriage prevalence in those hospitalised with ARI was 45.5% [95% CI 42.1-48.9] (389/855) and 43.8% of hospital attendees reported antibiotic use prior to admission. We did not identify significant associations with carriage of any serotypes in those with ARI. CONCLUSIONS: Pneumococcal serotypes 5 and 1 are associated with high invasive potential. Despite high community pneumococcal carriage, pre-hospital antibiotic usage likely reduces pneumococcal detection among children admitted in this setting and further research is needed to investigate serotypes associated with ARI. Data from this study can guide future preventative vaccination strategies in Malawi.

Quality of antibody responses by adults and young children to 13-valent pneumococcal conjugate vaccination and Streptococcus pneumoniae colonisation.

Childhood pneumococcal conjugate vaccine (PCV) protects against invasive pneumococcal disease caused by vaccine-serotype (VT) Streptococcus pneumoniae by generating opsonophagocytic anti-capsular antibodies, but how vaccination protects against and reduces VT carriage is less well understood. Using serological samples from PCV-vaccinated Malawian individuals and a UK human challenge model, we explored whether antibody quality (IgG subclass, opsonophagocytic killing, and avidity) is associated with protection from carriage. Following experimental challenge of adults with S. pneumoniae serotype 6B, 3/21 PCV13-vaccinees were colonised with pneumococcus compared to 12/24 hepatitis A-vaccinated controls; PCV13-vaccination induced serotype-specific IgG, IgG1, and IgG2, and strong opsonophagocytic responses. However, there was no clear relationship between antibody quality and protection from carriage or carriage intensity after vaccination. Similarly, among PCV13-vaccinated Malawian infants there was no relationship between serotype-specific antibody titre or quality and carriage through exposure to circulating serotypes. Although opsonophagocytic responses were low in infants, antibody titre and avidity to circulating serotypes 19F and 6A were maintained or increased with age. These data suggest a complex relationship between antibody-mediated immunity and pneumococcal carriage, and that PCV13-driven antibody quality may mature with age and exposure.

A pragmatic health centre-based evaluation comparing the effectiveness of a PCV13 schedule change from 3+0 to 2+1 in a high pneumococcal carriage and disease burden setting in Malawi: a study protocol.

INTRODUCTION: Streptococcus pneumoniae (the pneumococcus) is commonly carried as a commensal bacterium in the nasopharynx but can cause life-threatening disease. Transmission occurs by human respiratory droplets and interruption of this process provides herd immunity. A 2017 WHO Consultation on Optimisation of pneumococcal conjugate vaccines (PCV) Impact highlighted a substantial research gap in investigating why the impact of PCV vaccines in low-income countries has been lower than expected. Malawi introduced the 13-valent PCV (PCV13) into the national Expanded Programme of Immunisations in 2011, using a 3+0 (3 primary +0 booster doses) schedule. With evidence of greater impact of a 2+1 (2 primary +1 booster dose) schedule in other settings, including South Africa, Malawi's National Immunisations Technical Advisory Group is seeking evidence of adequate superiority of a 2+1 schedule to inform vaccine policy. METHODS: A pragmatic health centre-based evaluation comparing impact of a PCV13 schedule change from 3+0 to 2+1 in Blantyre district, Malawi. Twenty government health centres will be randomly selected, with ten implementing a 2+1 and 10 to continue with the 3+0 schedule. Health centres implementing 3+0 will serve as the direct comparator in evaluating 2+1 providing superior direct and indirect protection against pneumococcal carriage. Pneumococcal carriage surveys will evaluate carriage prevalence among children 15-24 months, randomised at household level, and schoolgoers 5-10 years of age, randomly selected from school registers. Carriage surveys will be conducted 18 and 33 months following 2+1 implementation. ANALYSIS: The primary endpoint is powered to detect an effect size of 50% reduction in vaccine serotype (VT) carriage among vaccinated children 15-24 months old, expecting a 14% and 7% VT carriage prevalence in the 3+0 and 2+1 arms, respectively. ETHICS AND DISSEMINATION: The study has been approved by the Malawi College of Medicine Research Ethics Committee (COMREC; Ref: P05.19.2680), the University College London Research Ethics Committee (Ref: 8603.002) and the University of Liverpool Research Ethics Committee (Ref: 5439). The results from this study will be actively disseminated through manuscript publications and conference presentations. TRIAL REGISTRATION NUMBER: NCT04078997.

Evaluation of Pneumococcal Serotyping of Nasopharyngeal-Carriage Isolates by Latex Agglutination, Whole-Genome Sequencing (PneumoCaT), and DNA Microarray in a High-Pneumococcal-Carriage-Prevalence Population in Malawi.

Accurate assessment of the serotype distribution associated with pneumococcal colonization and disease is essential for evaluating and formulating pneumococcal vaccines and for informing vaccine policy. For this reason, we evaluated the concordance between pneumococcal serotyping results by latex agglutination, whole-genome sequencing (WGS) with PneumoCaT, and DNA microarray for samples from community carriage surveillance in Blantyre, Malawi. Nasopharyngeal swabs were collected according to WHO recommendations between 2015 and 2017 by using stratified random sampling among study populations. Participants included healthy children 3 to 6 years old (vaccinated with the 13-valent pneumococcal conjugate vaccine [PCV13] as part of the Expanded Program on Immunization [EPI]), healthy children 5 to 10 years old (age-ineligible for PCV13), and HIV-infected adults (18 to 40 years old) on antiretroviral therapy (ART). For phenotypic serotyping, we used a 13-valent latex kit (Statens Serum Institut [SSI], Denmark). For genomic serotyping, we applied the PneumoCaT pipeline to whole-genome sequence libraries. For molecular serotyping by microarray, we used the BUGS Bioscience Senti-SP microarray. A total of 1,347 samples were analyzed. Concordance was 90.7% (95% confidence interval [CI], 89.0 to 92.2%) between latex agglutination and PneumoCaT, 95.2% (95% CI, 93.9 to 96.3%) between latex agglutination and the microarray, and 96.6% (95% CI, 95.5 to 97.5%) between the microarray and PneumoCaT. By detecting additional vaccine serotype (VT) pneumococci carried at low relative abundances (median, 8%), the microarray increased VT detection by 31.5% over that by latex serotyping. To conclude, all three serotyping methods were highly concordant in identifying dominant serotypes. Latex serotyping is accurate in identifying vaccine serotypes and requires the least expertise and resources for field implementation and analysis. However, WGS, which adds population structure, and microarray, which adds multiple-serotype carriage, should be considered at regional reference laboratories for investigating the importance of vaccine serotypes at low relative abundances in transmission and disease.