Emergence of nonvaccine Streptococcus pneumoniae following a 13-valent pneumococcal conjugate vaccine and schedule change in Mozambique: implications for a national immunization strategy

Emergence of nonvaccine Streptococcus pneumoniae following a 13-valent pneumococcal conjugate vaccine and schedule change in Mozambique: implications for a national immunization strategy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Emergence of nonvaccine Streptococcus pneumoniae following a 13-valent pneumococcal conjugate vaccine and schedule change in Mozambique: implications for a national immunization strategy Aquino Albino Nhantumbo, Linda Gouveia, Goitom Weldegebriel, Carlos Abel Funzamo, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7722758/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 24 You are reading this latest preprint version Abstract Background In Mozambique, the introduction of a 13-valent pneumococcal conjugate vaccine (PCV13) using a two plus one schedule began in September 2019. However, the replacement of serotypes with non-PCV types remains a concern worldwide following widespread vaccination. We aim to describe the epidemiology of non-PCV13 types among children under 5 years of age with pneumococcal meningitis four years after PCV13 introduction in Mozambique. Methods Between January 2020 and December 2023, a total of 2,014 cerebrospinal fluid (CSF) samples were collected from eligible children as part of the meningitis surveillance system in Mozambique, 103 (5.1%) of which tested positive for S. pneumoniae after triplex qPCR. Among these, 86 (83.5%) samples met the quality criteria for serotyping and were subsequently analysed using sequential quadriplex qPCR. Results Among children under 2 years of age, 84.8% of the S. pneumoniae- positive samples belonged to non-PCV13 serotypes, with serotype 15B/C (31.0%) being the most prevalent, followed by 12F/12A/12B/44/46 (12.1%), 38B (10.6%), and 8 (7.6%). Serotypes 15B/C, 12F/12A/12B/44/46, 38, 8, and 1 were predominant in the northern region of the country (Nampula), whereas serotypes 15B/C and 12F/12A/12B/44/46 were most common in the central region (Sofala). The serotypes coverage of the PCV13, PCV15 and PCV20 vaccine formulations were 20.0%, 26.3% and 75.8%, respectively. Conclusion PCV13 vaccines save many lives in Mozambique. However, they do not seem to be adequate in addresing some of the highly prevailing non-PCV13 serotypes (15B/C and 12F/12A/12B/44/46). Therefore, we suggest that the country's National Immunization Technical Advisory Group (NITAG) review new data and advice on switching to extended valency vaccines such as PCV20. Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Streptococcus pneumoniae (pneumococcus) remains one of the most important causes of life-threatening infections worldwide, with the highest burden in sub-Saharan Africa and Asia [ 1 – 5 ]. Mortality due to invasive pneumococcal disease (IPD) is still high and was estimated to have caused 225,000 deaths in children under 5 years of age worldwide in 2019 [ 1 ]. Meningitis is the most severe clinical presentation of pneumococcal disease, with an estimated burden of 44500 deaths among children under 5 years in 2019 [ 1 ] and a high case fatality ratio of up to 50% in low-income countries [ 1 – 6 ]. Vaccination using pneumococcal conjugate vaccines (PCVs) is the most crucial preventive measure for reducing the burden of pneumococcal diseases and is currently used in most countries. The introduction of PCVs into the Expanded Program on Immunization (EPI) has led to a reduction of over 80% in the global burden of IPDs caused by vaccine-type S. pneumoniae serotypes [ 1 , 5 , 7 – 13 ]. In addition, evidence from a variety of settings has demonstrated that PCVs are highly protective against vaccine-type IPD and provide substantial indirect protection. As a result, their use has been recommended by the WHO for infants worldwide [ 1 , 2 , 8 – 16 ]. However, despite the significant decline in vaccine-type IPD, the emergence and increasing incidence of disease caused by non-PCV serotypes may undermine the overall benefits of vaccination. This serotype replacement poses a growing public health concern in many countries following the introduction of PCV10 or PCV13 vaccines [ 14 – 16 ]. In particular, an increase in nonvaccine serotypes, such as 8, 12F,15A/B/C, 16F,17F, 22F, 23A/B, 6C,10A, 24F, 6C, 35B, 10A, 9 N, 11A, 10F, 9F, and 20, has been reported [ 14 – 16 ]. Therefore, higher-valence vaccines have been developed. The 15-valent PCV (PCV15; Vaxneuvance, Merck Sharp & Dohme, Whitehouse Station, NJ, USA) includes two additional serotypes (22F and 33F) beyond those covered by PCV13 (1, 3, 4, 5, 6A, 6B, 7F, 9 V, 14, 18C, 19A, 19F, and 23F) and has recently been recommended for pediatric use in the USA [ 17 ]. The 20-valent PCV (PCV20; Prevnar 20, Pfizer, NY, USA), which adds serotypes 8, 10A, 11A, 12F, and 15B/C to those in PCV15 to make it PCV20, is currently used only in adults [ 17 , 18 ]. In Mozambique, the PCV10 (Synflorix, Glaxo-Smith-Kline Vaccine) formulation vaccine was the first to be introduced into EPI in April 2013 as a three-dose schedule without a booster (3p + 0 schedule) administered at 6, 10, and 14 weeks of age [ 10 ]. Shortly after its introduction, a 97.5% reduction in pneumococcal meningitis was observed [ 10 ]. In December 2017, Mozambique switched from PCV10 to PCV13 through a phased rollout starting in the northern and central regions of the country, which was completed nationally by May 2019; however, the use of the new vaccination schedule of two primary doses given at ages 2 and 4 months with a booster at 9 months (2p + 1 schedule) began in September 2019. Despite these efforts, data on the epidemiology of nonvaccine serotypes of S. pneumoniae following PCV introduction in Mozambique remain scarce. This study aimed to assess the epidemiology of non-PCV13 pneumococcal serotypes causing acute bacterial meningitis in children under five years of age four years after PCV13 vaccine introduction using a 2p + 1 schedule in Mozambique. Methods Study setting and population A retrospective cross-sectional study was conducted using data from the routine sentinel surveillance system for paediatric bacterial meningitis in Mozambique. Since 2013, the Instituto Nacional de Saúde (INS) of Mozambique has conducted ongoing hospital-based surveillance for vaccine-preventable bacterial meningitis among children under five years of age at the three largest referral hospitals in the Mozambique-Maputo Central Hospital (MCH), Beira Central Hospital (BCH), and Nampula Central Hospital (NCH) located in the southern, central, and northern regions of the country, respectively [ 19 ]. All three hospitals have been capacitated to provide several specialized healthcare services for all age groups and receive and treat all meningitis cases reported in hospitals within their respective catchment areas due to their capacity to manage meningitis cases. The catchment population for each hospital was determined on the basis of administrative regions within each province via projections from the 2017 census (population from 2020–2023). During this period, the population under 5 years of age served by each hospital was estimated at 553,793 for MCH, 510,154 for BCH and 671,322 for NCH (Supplementary Table S1 ) [ 20 ]. Clinical specimens were consecutively collected from children under 5 years of age with suspected meningitis and tested by culture with 5% sheep blood and chocolate agar plates [MAST, Merseyside, UK] at the three main referral hospitals (MCH, BCH and NCH) [ 19 , 20 ]. The study sample included all pneumococcal isolates stored in skim milk-tryptone-glucose-glycerol (STGG) medium as well as any residual CSF samples preserved at − 70°C at the INS of Mozambique. Pneumococcal meningitis was defined as the presence of S. pneumoniae detected in CSF using multiplex qPCR [ 21 ]. Detection of S. pneumoniae using qPCR The molecular diagnosis of S. pneumoniae was performed as part of the routine diagnostic procedures for meningitis within the national paediatric bacterial meningitis surveillance system implemented by the INS. Diagnosis was routinely performed via triplex real-time PCR (qPCR) [ 22 ]. This assay employs a specific set of specific primers and probes in a single multiplex reaction using FAM, Cy5, and HEX fluorophores to target the sodC gene (Cu-Zn superoxide dismutase) for N. meningitidis , the lyt A gene (autolysin) for S. pneumoniae and the hpd gene (protein D) for H. influenzae , respectively [ 22 ]. The standard reference strains used as positive controls included S. pneumoniae ATCC 49619, H. influenzae ATCC 49247 and N. meningitidis ATCC13077. No-template controls (NTCs) were included in each reaction as negative controls. All samples with a cycle threshold (ct) value ≤ 35 were considered positive [ 22 ]. Determination of Pneumococcal Serotypes All pneumococcal isolates and S. pneumoniae PCR - positive CSF samples collected between January 2020 and December 2023 were serotyped at the INS in Mozambique using real-time PCR assays performed in 12 quadriplex reactions. These assays target 48 serotypes, including serotypes not covered by previous methods, such as 22F, 22A, 6AB, 6AB, 6BD, 31, 7C/7B1, 34, 38, 28A/28F, 37, 11B/11C, 10F, and 35A [ 23 ]. Pneumococcal meningitis cases were categorized as follows: PCV13 serotypes (1, 4, 5, 6B, 7F, 9 V, 14, 18C, 19F, 23F 3, 6A, and 19A), PCV15 serotypes (all PCV13 serotypes plus 22F and 33F), PCV20 serotypes (all PCV15 serotypes plus 8, 10A, 11A, 12F, and 15B), and non-PCV13 serotypes (all other serotypes, including nontypable pneumococci, and PCR negative for the 40 serotypes included in the multiplex real-time PCR). Samples with low DNA concentrations (Ct > 35) and insufficient CSF volume were classified as missing. Statistical analysis Statistical analysis was performed using the R statistical software version 4.1.1 (Vienna, Austria). Categorical variables are reported as proportions and were compared using the Pearson chi-square test, as appropriate. P values < 0.05 were considered statistically significant. Ethics statement Ethical approval for this study was given by the Mozambican National Bioethics Committee Ref #: 180/CNBS/20/IRB00002657 and the study was performed in accordance with the Declaration of Helsinki.Verbal consent was obtained from the parents of all participating children’s parents/caregivers during routine medical care for paediatric bacterial meningitis in Mozambique [ 19 ]. Results General characteristics of the study participants During the observation period (January 2020 to December 2023), a total of 2120 children under 5 years of age with a clinical diagnosis of meningitis were enrolled across the three sentinel hospitals; CSF samples were successfully collected, and 2,014 (95.0%) CSF samples were collected (Fig. 1 ). The median age was 9 months (interquartile range (IQR) 4–24 months), and 55.7% (1121/2014) were male. By site, 85.5% were from NCH, 8.2% were from BCH, and 6.3% were from MCH (Fig. 1 , Table 1 ). Over the four years following PCV13 introduction, S. pneumoniae was detected via PCR in 103 of the 2,014 CSF samples (5.1%) from children under 5 years of age with suspected meningitis.. Among these samples, 86 (83.5%) with Ct values ≤ 35 were subjected to serotyping. The age distribution of the PCR-positive cases mirrored that of the overall cohort, with the majority of S. pneumoniae- positive children aged less than 12 months following the same pattern as the enrolled children (Table 1 ). Table 1 Distribution of enrolled patients, CSF PCR results and typeable samples by age, sex and region (2020–2023). Characteristics Total (n = 2,014) S. pneumoniae (n = 103) Children with typable CSF (n = 86) Age in months, median (IQR) 9 (4–24) 11 (5–24) 11,5 (5–27) Age group 0–11 months 1178 (58.5%) 55 (53.4%) 43 (50.0%) 12–23 months 262 (13.0%) 18 (17.5%) 17 (19.8%) 24–59 months 574 (28.5%) 30 (29.1%) 26 (30.2%) Gender Male 1121 (55.7%) 57 (55.3%) 47 (54.7%) Female 893 (44.3%) 46 (44.7%) 39 (45.3%) Study sites NCH (northern) 1722 (85.5%) 89 (86.4%) 75 (87.2%) BCH (central) 166 (8.2%) 12 (11.7%) 10 (11.6%) MCH (southern) 126 (6.3%) 2 (1.9%) 1 (1.2%) Serotype distribution and vaccine formulation coverage Among the 95 samples subjected to serotyping, 86 (90.5%) presented a specific serotype, whereas the remaining nine (9.5%) samples were PCR negative for the 40 serotypes included in the multiplex and were classified as nontypable pneumococci (NTPs), as shown in Fig. 1 . Four years after PCV13 introduction, the most predominant strains identified were 15B/C (28.4%, 27/95), 12F/12A/12B/44/46 (14.7%, 14/95), 1 (9.5%, 9/95), 38 (8.4%, 8/95), 8 (6.3%, 6/95), 16F (3.2%, 3/95), 24F/24A/24B (3.2%, 3/95), 35B/18F (3/95), 14 (3.2%, 3/95), 9 V/9A (2.1%, 2/95), 19F (2.1%, 2/95), 23F (1.1%, 1/95), 4 (1.1%, 1/95), 18A/18B/18C/18F (1.1%, 1/95), 22F/22A (1.1%, 1/95), 7C/7B2 (1.1%, 1/95), and 17F (1.1%, 1/95) (Fig. 2 ). When stratified by age group, children under 2 years (n = 66) harbored predominantly non-PCV13 serotypes (84.8%, 56/66). The five most common serotypes were 15B/C (31.0%), followed by 12F/12A/12B/44/46 (12.1%), 38B (10.6%), 8 (11.1%), and 35B (4.5%), together accounting for 66.7% (44/66) of the isolates (Fig. 3 ). The PCV13 serotypes represented 20% (n = 19/95) of all cases, including serotypes 1, 14, 9 V/9A, 23F, and 19F. Among children aged ≥ 2 years, the most prevalent serotypes were 15B/C (20.7%, 6/29), 12F/12A/12B/44/46 (20.7%, 6/29), and 1 (20.7%, 6/29), followed by 24F/22A/222B (6.9%) (Fig. 3 ). These four types accounted for 69.0% (20/29) of all the serotypes. The less frequent serotypes (3.5% each) included 16F, 38, 8,19F, 9 V/9A and 18C/18F/18B/18A (Fig. 3 ). Moreover, the rates of vaccine coverage against the serotypes of S. pneumoniae causing meningitis among children under 5 years of age in Mozambique post-PCV13 introduction in Mozambique (2020–2023) were 20.0% (19/95), 26.3% (25/95), and 75.8% (72/95) for PCV-13, PCV-15, and PCV-20, respectively (Fig. 2 ). In terms of geographic distribution, serotypes 15B/C, 12F/12A/12B/44/46, 38, 8, and 1 were the most predominant in the northern region of the country (Nampula) after PCV13 introduction, whereas serotypes 15B/C and 12F/12A/12B/44/46 predominated in the central region (Sofala) (Fig. 4 ). In the southern region of the country (Maputo), only one case of serotype 1 and another nontypeable case were recorded. Discussion In this study, we reported for the first time the prevalence of non-PCV13 pneumococcal serotypes causing acute bacterial meningitis in children under 5 years four years after the introduction of the PCV13 2p + 1 schedule in Mozambique. Our findings revealed a high prevalence of non-PCV13 serotypes (80.0%) compared with the 18.8% reported in our earlier surveillance of confirmed pediatric pneumococcal meningitis [ 10 ]. Consequently, only 20% of cases were attributable to PCV13 serotypes, indicating that the current vaccine formulation provides limited coverage in Mozambique. These results are consistent with post-PCV introduction data from other previously reported findings in many African countries [ 24 – 27 ], Europe [ 28 , 29 ], Asian countries [ 30 , 32 ], Latin America [ 32 , 33 ], and North America [ 34 ], all of which reported low residual prevalence (~ 28%) of vaccine-type serotypes following PCV implementation. In a 2017 study of S. pneumoniae isolates from children with acute bacterial meningitis in Mozambique, Nhantumbo et al. [ 10 ] reported that PCV13 serotypes 1, 5, 6A, 6B, 23F, 14, 9 V/9A, 4, 3, 19A, and 18A/18B/18C/18F accounted for 81.2% of cases in children under five years of age. Four years later, the present study did not detect several of these serotypes, namely, 5, 3, 6A, 6B, and 19A, highlighting their near elimination. These findings are consistent with previous observations in many African countries, underscoring the impact of immunization strategies in protecting children under 5 years of age from invasive pneumococcal diseases caused by vaccine serotypes [ 24 – 27 ]. In the present study, the most prevalent non-PCV13 replacement serotypes in Mozambique were 15B/C, 12F/12A/12B/44/468, 22F/22A, 38, 8, 35B, 24F/24A/24B, and 16F. Together, these serotypes accounted for 84.8% of all S. pneumoniae isolates causing ABM in children aged < 2 years. This marked increase in nonvaccine serotypes reflects the serotype replacement phenomenon observed after PCV introduction and aligns with trends reported in other countries following the rollout of pneumococcal conjugate vaccines [ 35 – 38 ]. Overall, serotypes 8, 12F, 15B, 15A, 22F, 33, 38, 8, 35B, 24F/24A/24B, and 16F have emerged as the predominant replacement serotypes recovered from pediatric invasive pneumococcal disease cases worldwide following PCV implementation in both low- and high-income countries [ 35 – 42 ]. This global serotype replacement phenomenon, characterized by the increase in nonvaccine types after the introduction of PCVs, has been repeatedly reported across diverse settings [ 35 – 42 ]. Another notable finding in our study was that 20.7% of residual serotype 1 cases occurred in children under 2 years of age. However, all of these affected children were unvaccinated (Supplementary Table S2). In many countries, the introduction of PCVs using a 2p + 1 schedule has led to a substantial reduction in disease caused by vaccine-type serotypes, including serotype 1 [ 36 ]. The geographic distribution of S. pneumomiae serotypes in Mozambique revealed slight variations. In the northern region of the country (Nampula Province), the predominant serotypes were 15B/C, 12F/12A/12B/44/46, 38, 8, 1, and 14, whereas in the central region of the country (Sofala Province), serotypes 15B/C and 12F/12A/12B/44/46 predominated. In the southern region of the country, only serotype 1 was detected. The frequency and interregional spread of these serotypes can be affected by several factors, such as population mobility, vaccination coverage, public health policies, and environmental conditions [ 43 – 47 ]. Regarding PCV13 serotype coverage, our findings indicate that this vaccine formulation covers only 20.0% (19/95) of the serotypes identified. These results are similar to those reported in several countries in Africa, the Americas, Europe, and Asia, where post-PCV13 coverage rates ranged from 10.0 to 26.4% [ 48 ]. In contrast, higher-valence vaccines, such as PCV20, which includes serotypes 22F, 33F, 8, 10A, 11A, 12F, and 15B/C, cover 75.0% (72/95) of the serotypes identified in our study. These findings are consistent with global data reporting coverage rates of 74.0% for PCV20 [ 48 ]. Nampula is one of the most populous provinces in the country, with a total population of 6.102.867 [ 20 ]. A poor resource setting in northern Mozambique, likely related to higher levels of social disadvantage, is associated with significantly higher rates of morbidity across a range of infectious diseases, including pneumococcal disease. Although PCV13 vaccine coverage was greater than 90% nationwide, in Nampula Province, PCV13 vaccine coverage for the 3rd dose decreased from 83.5 to 69.0% in 2020 and 2022, respectively. Increased vaccination coverage in the age groups implicated in disease transmission has been reported to generate indirect effects of vaccination programs at the population level [ 49 , 50 ]. With increased indirect effects, the reduction or elimination of transmission and IPD caused by vaccine serotypes can be achieved. Furthermore, the elimination of IPD caused by vaccine serotypes has been reported largely in settings that rapidly achieved very high coverage rates of PCV vaccines [ 51 ]. We acknowledge several limitations that may have influenced our results. First, the data were collected from the three largest hospitals in Mozambique, which may limit the representativeness of our findings. Second, our molecular serotyping method targets only 48 of the most common serotypes/serogroups, which likely explains the few untyped samples, most likely due to the absence of those specific serotypes in our multiplex PCR panel. Conclusion Our study provides, for the first time, evidence of a high prevalence of non-PCV13 serotypes causing pneumococcal meningitis among children under five years of age following the introduction of PCV in Mozambique. Although PCV13 vaccines save many lives in Mozambique, they do not seem to be adequate in addresing some of the highly prevailing non-PCV13 serotypes (15B/C and 12F/12A/12B/44/46). Therefore, we suggest that the country's NITAG reviews available new data and advise that switching to broader-spectrum formulations such as PCV20 may offer better coverage and disease prevention, while the development of a universal pneumococcal vaccine is still pending. Abbreviations ABM acute bacterial meningitis CSF cerebrospinal fluid Ct cycle threshold DNA deoxyribonucleic acid BCH Beira Central Hospital MCH Maputo Central Hospital NCH :Nampula Central Hospital INS Instituto Nacional de Saúde IPD invasive pneumococcal diseases IQR interquartile range qPCR multiplex qualitative polymerase chain reaction NITAG National Immunization Technical Advisory Group NRML National Reference Microbiology Laboratory NTS nontypable specimen NVT nonvaccine type PCV-13 thirteen-valent conjugate vaccine RRL Regional Reference Laboratory Declarations Consent for publication Our manuscript does not include any individual personal data; however, consent to participate was obtained from each participant as stated in the Ethics Statement section. Competing interests : The authors declare that they have no competing interests. Disclaimer The findings and conclusions in this publication are those of the authors and do not necessarily represent the official position of the World Health Organization or the U.S. Centers for Disease Control and Prevention. Informed Consent Statement : Our manuscript does not include any individual personal data; however, consent to participate was obtained from each participant as stated in the Ethics Statement section. The study was performed in accordance with the Declaration of Helsinki. Funding: Gavi, The Vaccine Alliance, reference number: MOZ-HSS-2-INS; WHO Reference: 2014405143-0, creation DFC to support the HIB & Surveillance System. Author Contribution E.S.G., I.d.F., A.E.A., A.A.N., C.E.C., Z.C., R.K., E.R.C., L.d.G., E.V.Z., P.I.M., E.F.S., VC., C.D., M.V.N., C.A.F., A.M., A.C.d.F.N.M., and G.W. worked together on study conceptualization, data collection, supervision, and project administration. A.A.N., L.d.G., E.S.G., I.d.F., A.M.M., C.D., and V.C. performed the molecular experiments. E.S.G., R.M., R.K., I.d.F., A.E.A., G.W., and A.A.N. performed the data analysis and validation. A.A.N., I.d.F. and E.S.G. wrote the original draft of the manuscript. I.d.F., E.S.G., A.A.N., C.D., P.I.M., V.C., C.E.C., A.M.M., A.C.F.N.M., A.N.T., G.W., L.d.G., and C.E.C. interpreted the data and wrote the manuscript. All the authors have read and agreed to the published version of the manuscript. Acknowledgements: We thank Marcelino of the Bacteriology Laboratory of the Nampula Central Hospital (NCH), Timótio Bejamim and Joaquim João Marcos of the Bacteriology Laboratory of the Beira Central Hospital (BCH), and Célio Conjo and Calvina Langa of the Bacteriology Laboratory of the Maputo Central Hospital (MCH) for culturing and identifying the bacterial isolates. 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Serotype distribution of remaining invasive pneumococcal disease after extensive use of ten-valent and 13-valent pneumococcal conjugate vaccines (the PSERENADE project): a global surveillance analysis [published correction appears in Lancet Infect Dis. 2025;25(2):e68. doi: 10.1016/S1473-3099(25)00002-7.]. Lancet Infect Dis . 2025;25(4):445–456. 10.1016/S1473-3099(24)00588-7 Fine P, Eames K, Heymann DL. Herd Immunity: A Rough Guide. Clin Infect Dis. 2011;52:911–6. 10.1093/cid/cir007 . Halloran ME, Struchiner CJ, Longini IM. Study Designs for Evaluating the Efficacy and Effectiveness of Vaccines. Am J Epidemiol. 1997;146:789–803. 10.1093/oxfordjournals.aje.a009196 . Tsaban G, Ben-Shimol S. Indirect (herd) protection, following pneumococcal conjugated vaccines introduction: A systematic review of the literature. Vaccine. 2017;35:2882–91. 10.1016/j.vaccine.2017.04.032 . Additional Declarations No competing interests reported. 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01:20:33","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":152109,"visible":true,"origin":"","legend":"","description":"","filename":"74559c9f57154f2aba53e40f327dfd471structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7722758/v1/13e80bae5c8ea5794571a992.xml"},{"id":95223769,"identity":"b3a8fcbf-1389-4bd9-9f9c-d9f2373a9b79","added_by":"auto","created_at":"2025-11-05 16:22:47","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":171310,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7722758/v1/0327f6cd041f77f7f9b72923.html"},{"id":95064010,"identity":"9b985fcf-1855-40bb-af8c-8066c684ac77","added_by":"auto","created_at":"2025-11-04 01:20:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":35308,"visible":true,"origin":"","legend":"\u003cp\u003eStudy profile during the observation period from January 2020 to December 31, 2023. The flow chart depicts the total number of suspected acute bacterial meningitis cases among children under five years of age, along with the counts of CSF samples collected and tested during this period. Abbreviations. \u003cstrong\u003eABM: \u003c/strong\u003eacute bacterial meningitis\u003cstrong\u003e; BCH: \u003c/strong\u003eBeira Central Hospital; \u003cstrong\u003eCSF: \u003c/strong\u003ecerebrospinal fluid\u003cstrong\u003e; DNA: \u003c/strong\u003edeoxyribonucleic acid;\u003cstrong\u003e INS: \u003c/strong\u003eInstituto Nacional de Saúde;\u003cstrong\u003eMCH: \u003c/strong\u003eMaputo Central Hospital\u003cstrong\u003e; NCH: \u003c/strong\u003eNampula Central Hospital;\u003cstrong\u003eNMRL:\u003c/strong\u003e National Reference Microbiology Laboratory;\u003cstrong\u003e NTS: \u003c/strong\u003enontypable specimen;\u003cstrong\u003e PCR: \u003c/strong\u003epolymerase chain reaction;\u003cstrong\u003ePCV13: \u003c/strong\u003e13-valent pneumococcal conjugate vaccine.\u003c/p\u003e","description":"","filename":"Onlinefloatimage110.png","url":"https://assets-eu.researchsquare.com/files/rs-7722758/v1/1b6cd03650a612b58672dd72.png"},{"id":95222805,"identity":"8f0bb1db-f63a-49aa-91db-3418ba4b2ad8","added_by":"auto","created_at":"2025-11-05 16:21:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":158702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution and coverage rates of local \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eserotypes according to vaccine type (PCV-13, PCV-15 and PCV20) between 2020 and 2023. \u003c/strong\u003eEach bar represents the relative frequency of each serotype of \u003cem\u003eS. pneumoniae\u003c/em\u003e. The values in the arrows above the bars depict the coverage of the \u003cem\u003eS. pneumoniae\u003c/em\u003eserotype by PCV13, PCV15, and PCV20. Abbreviations: \u003cstrong\u003eNTS,\u003c/strong\u003e nontypable sample.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7722758/v1/9763f89f75d315c929a67c47.png"},{"id":95223274,"identity":"abb274d8-43eb-4008-bf3f-ecfb399afb51","added_by":"auto","created_at":"2025-11-05 16:21:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":77553,"visible":true,"origin":"","legend":"\u003cp\u003eProportions of pneumococcal meningitis isolates stratified by pneumococcal serotype and age. Each bar represents the relative frequency of each serotype of \u003cem\u003eS. pneumoniae\u003c/em\u003e. Abbreviations. \u003cstrong\u003eNTS: \u003c/strong\u003enontypable sample.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7722758/v1/24aa434872bfb2beeb263222.png"},{"id":95222968,"identity":"52659405-a8bb-4fe7-a43e-5d149beb04bb","added_by":"auto","created_at":"2025-11-05 16:21:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":379299,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of serotypes of \u003cem\u003eS. pneumoniae\u003c/em\u003e stratified by region between January 2020 and March 2023. Figure depicts the distribution of \u003cem\u003eS. pneumoniae serotypes\u003c/em\u003e in southern (HCM), central (HCB) and northern (HCN) Mozambique. Each color represents one serotype. Abbreviations. \u003cstrong\u003eBCH: \u003c/strong\u003eBeira Central Hospital; \u003cstrong\u003eMCH: \u003c/strong\u003eMaputo Central Hospital\u003cstrong\u003e; NCH: \u003c/strong\u003eNampula Central Hospital; \u003cstrong\u003eNTS\u003c/strong\u003e: non-typable specimen\u003c/p\u003e","description":"","filename":"floatimage47.png","url":"https://assets-eu.researchsquare.com/files/rs-7722758/v1/9c61b9640099d512eebd2699.png"},{"id":95229951,"identity":"9fa8f6a8-621a-4ff1-ba17-b4384bcb35f5","added_by":"auto","created_at":"2025-11-05 16:36:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1572161,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7722758/v1/7944c8e2-81a2-44fc-8151-a79c9ad7e6af.pdf"},{"id":95064012,"identity":"e19f2915-09b1-4311-ba18-1bc2d7f76223","added_by":"auto","created_at":"2025-11-04 01:20:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19111,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7722758/v1/8c1fa6b76eed020873985ea1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Emergence of nonvaccine Streptococcus pneumoniae following a 13-valent pneumococcal conjugate vaccine and schedule change in Mozambique: implications for a national immunization strategy","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e (pneumococcus) remains one of the most important causes of life-threatening infections worldwide, with the highest burden in sub-Saharan Africa and Asia [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Mortality due to invasive pneumococcal disease (IPD) is still high and was estimated to have caused 225,000 deaths in children under 5 years of age worldwide in 2019 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Meningitis is the most severe clinical presentation of pneumococcal disease, with an estimated burden of 44500 deaths among children under 5 years in 2019 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and a high case fatality ratio of up to 50% in low-income countries [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Vaccination using pneumococcal conjugate vaccines (PCVs) is the most crucial preventive measure for reducing the burden of pneumococcal diseases and is currently used in most countries. The introduction of PCVs into the Expanded Program on Immunization (EPI) has led to a reduction of over 80% in the global burden of IPDs caused by vaccine-type \u003cem\u003eS. pneumoniae\u003c/em\u003e serotypes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11 CR12\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, evidence from a variety of settings has demonstrated that PCVs are highly protective against vaccine-type IPD and provide substantial indirect protection. As a result, their use has been recommended by the WHO for infants worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13 CR14 CR15\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, despite the significant decline in vaccine-type IPD, the emergence and increasing incidence of disease caused by non-PCV serotypes may undermine the overall benefits of vaccination. This serotype replacement poses a growing public health concern in many countries following the introduction of PCV10 or PCV13 vaccines [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn particular, an increase in nonvaccine serotypes, such as 8, 12F,15A/B/C, 16F,17F, 22F, 23A/B, 6C,10A, 24F, 6C, 35B, 10A, 9 N, 11A, 10F, 9F, and 20, has been reported [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, higher-valence vaccines have been developed. The 15-valent PCV (PCV15; Vaxneuvance, Merck Sharp \u0026amp; Dohme, Whitehouse Station, NJ, USA) includes two additional serotypes (22F and 33F) beyond those covered by PCV13 (1, 3, 4, 5, 6A, 6B, 7F, 9 V, 14, 18C, 19A, 19F, and 23F) and has recently been recommended for pediatric use in the USA [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The 20-valent PCV (PCV20; Prevnar 20, Pfizer, NY, USA), which adds serotypes 8, 10A, 11A, 12F, and 15B/C to those in PCV15 to make it PCV20, is currently used only in adults [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn Mozambique, the PCV10 (Synflorix, Glaxo-Smith-Kline Vaccine) formulation vaccine was the first to be introduced into EPI in April 2013 as a three-dose schedule without a booster (3p\u0026thinsp;+\u0026thinsp;0 schedule) administered at 6, 10, and 14 weeks of age [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Shortly after its introduction, a 97.5% reduction in pneumococcal meningitis was observed [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In December 2017, Mozambique switched from PCV10 to PCV13 through a phased rollout starting in the northern and central regions of the country, which was completed nationally by May 2019; however, the use of the new vaccination schedule of two primary doses given at ages 2 and 4 months with a booster at 9 months (2p\u0026thinsp;+\u0026thinsp;1 schedule) began in September 2019. Despite these efforts, data on the epidemiology of nonvaccine serotypes of \u003cem\u003eS. pneumoniae\u003c/em\u003e following PCV introduction in Mozambique remain scarce. This study aimed to assess the epidemiology of non-PCV13 pneumococcal serotypes causing acute bacterial meningitis in children under five years of age four years after PCV13 vaccine introduction using a 2p\u0026thinsp;+\u0026thinsp;1 schedule in Mozambique.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy setting and population\u003c/h2\u003e\u003cp\u003eA retrospective cross-sectional study was conducted using data from the routine sentinel surveillance system for paediatric bacterial meningitis in Mozambique. Since 2013, the Instituto Nacional de Sa\u0026uacute;de (INS) of Mozambique has conducted ongoing hospital-based surveillance for vaccine-preventable bacterial meningitis among children under five years of age at the three largest referral hospitals in the Mozambique-Maputo Central Hospital (MCH), Beira Central Hospital (BCH), and Nampula Central Hospital (NCH) located in the southern, central, and northern regions of the country, respectively [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. All three hospitals have been capacitated to provide several specialized healthcare services for all age groups and receive and treat all meningitis cases reported in hospitals within their respective catchment areas due to their capacity to manage meningitis cases. The catchment population for each hospital was determined on the basis of administrative regions within each province via projections from the 2017 census (population from 2020\u0026ndash;2023). During this period, the population under 5 years of age served by each hospital was estimated at 553,793 for MCH, 510,154 for BCH and 671,322 for NCH (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eClinical specimens were consecutively collected from children under 5 years of age with suspected meningitis and tested by culture with 5% sheep blood and chocolate agar plates [MAST, Merseyside, UK] at the three main referral hospitals (MCH, BCH and NCH) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The study sample included all pneumococcal isolates stored in skim milk-tryptone-glucose-glycerol (STGG) medium as well as any residual CSF samples preserved at \u0026minus;\u0026thinsp;70\u0026deg;C at the INS of Mozambique.\u003c/p\u003e\u003cp\u003ePneumococcal meningitis was defined as the presence of \u003cem\u003eS. pneumoniae\u003c/em\u003e detected in CSF using multiplex qPCR [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of\u003c/b\u003e \u003cb\u003eS. pneumoniae\u003c/b\u003e \u003cb\u003eusing qPCR\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe molecular diagnosis of \u003cem\u003eS. pneumoniae\u003c/em\u003e was performed as part of the routine diagnostic procedures for meningitis within the national paediatric bacterial meningitis surveillance system implemented by the INS. Diagnosis was routinely performed via triplex real-time PCR (qPCR) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This assay employs a specific set of specific primers and probes in a single multiplex reaction using FAM, Cy5, and HEX fluorophores to target the \u003cem\u003esodC\u003c/em\u003e gene (Cu-Zn superoxide dismutase) for \u003cem\u003eN. meningitidis\u003c/em\u003e, the \u003cem\u003elyt\u003c/em\u003eA gene (autolysin) for \u003cem\u003eS. pneumoniae\u003c/em\u003e and the \u003cem\u003ehpd\u003c/em\u003e gene (protein D) for \u003cem\u003eH. influenzae\u003c/em\u003e, respectively [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The standard reference strains used as positive controls included \u003cem\u003eS. pneumoniae\u003c/em\u003e ATCC 49619, \u003cem\u003eH. influenzae\u003c/em\u003e ATCC 49247 and \u003cem\u003eN. meningitidis\u003c/em\u003e ATCC13077. No-template controls (NTCs) were included in each reaction as negative controls. All samples with a cycle threshold (ct) value\u0026thinsp;\u0026le;\u0026thinsp;35 were considered positive [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDetermination of Pneumococcal Serotypes\u003c/strong\u003e\u003cp\u003eAll pneumococcal isolates and \u003cem\u003eS. pneumoniae\u003c/em\u003e PCR\u003cem\u003e-\u003c/em\u003epositive CSF samples collected between January 2020 and December 2023 were serotyped at the INS in Mozambique using real-time PCR assays performed in 12 quadriplex reactions. These assays target 48 serotypes, including serotypes not covered by previous methods, such as 22F, 22A, 6AB, 6AB, 6BD, 31, 7C/7B1, 34, 38, 28A/28F, 37, 11B/11C, 10F, and 35A [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003c/p\u003e\u003cp\u003ePneumococcal meningitis cases were categorized as follows: PCV13 serotypes (1, 4, 5, 6B, 7F, 9 V, 14, 18C, 19F, 23F 3, 6A, and 19A), PCV15 serotypes (all PCV13 serotypes plus 22F and 33F), PCV20 serotypes (all PCV15 serotypes plus 8, 10A, 11A, 12F, and 15B), and non-PCV13 serotypes (all other serotypes, including nontypable pneumococci, and PCR negative for the 40 serotypes included in the multiplex real-time PCR). Samples with low DNA concentrations (Ct\u0026thinsp;\u0026gt;\u0026thinsp;35) and insufficient CSF volume were classified as missing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using the R statistical software version 4.1.1 (Vienna, Austria). Categorical variables are reported as proportions and were compared using the Pearson chi-square test, as appropriate. \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEthics statement\u003c/h3\u003e\n\u003cp\u003eEthical approval for this study was given by the Mozambican National Bioethics Committee Ref #: 180/CNBS/20/IRB00002657 and the study was performed in accordance with the Declaration of Helsinki.Verbal consent was obtained from the parents of all participating children\u0026rsquo;s parents/caregivers during routine medical care for paediatric bacterial meningitis in Mozambique [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eGeneral characteristics of the study participants\u003c/h2\u003e\u003cp\u003eDuring the observation period (January 2020 to December 2023), a total of 2120 children under 5 years of age with a clinical diagnosis of meningitis were enrolled across the three sentinel hospitals; CSF samples were successfully collected, and 2,014 (95.0%) CSF samples were collected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The median age was 9 months (interquartile range (IQR) 4\u0026ndash;24 months), and 55.7% (1121/2014) were male. By site, 85.5% were from NCH, 8.2% were from BCH, and 6.3% were from MCH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Over the four years following PCV13 introduction, \u003cem\u003eS. pneumoniae\u003c/em\u003e was detected via PCR in 103 of the 2,014 CSF samples (5.1%) from children under 5 years of age with suspected meningitis.. Among these samples, 86 (83.5%) with Ct values\u0026thinsp;\u0026le;\u0026thinsp;35 were subjected to serotyping. The age distribution of the PCR-positive cases mirrored that of the overall cohort, with the majority of \u003cem\u003eS. pneumoniae-\u003c/em\u003epositive children aged less than 12 months following the same pattern as the enrolled children (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDistribution of enrolled patients, CSF PCR results and typeable samples by age, sex and region (2020\u0026ndash;2023).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCharacteristics\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal (n\u0026thinsp;=\u0026thinsp;2,014)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eS. pneumoniae\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;103)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eChildren with typable CSF (n\u0026thinsp;=\u0026thinsp;86)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge in months, median (IQR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9 (4\u0026ndash;24)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11 (5\u0026ndash;24)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11,5 (5\u0026ndash;27)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u0026ndash;11 months\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1178 (58.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55 (53.4%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e43 (50.0%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u0026ndash;23 months\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e262 (13.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18 (17.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17 (19.8%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24\u0026ndash;59 months\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e574 (28.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30 (29.1%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26 (30.2%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1121 (55.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e57 (55.3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e47 (54.7%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e893 (44.3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46 (44.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39 (45.3%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStudy sites\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNCH (northern)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1722 (85.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e89 (86.4%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e75 (87.2%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBCH (central)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e166 (8.2%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12 (11.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10 (11.6%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCH (southern)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e126 (6.3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (1.9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (1.2%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSerotype distribution and vaccine formulation coverage\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAmong the 95 samples subjected to serotyping, 86 (90.5%) presented a specific serotype, whereas the remaining nine (9.5%) samples were PCR negative for the 40 serotypes included in the multiplex and were classified as nontypable pneumococci (NTPs), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Four years after PCV13 introduction, the most predominant strains identified were 15B/C (28.4%, 27/95), 12F/12A/12B/44/46 (14.7%, 14/95), 1 (9.5%, 9/95), 38 (8.4%, 8/95), 8 (6.3%, 6/95), 16F (3.2%, 3/95), 24F/24A/24B (3.2%, 3/95), 35B/18F (3/95), 14 (3.2%, 3/95), 9 V/9A (2.1%, 2/95), 19F (2.1%, 2/95), 23F (1.1%, 1/95), 4 (1.1%, 1/95), 18A/18B/18C/18F (1.1%, 1/95), 22F/22A (1.1%, 1/95), 7C/7B2 (1.1%, 1/95), and 17F (1.1%, 1/95) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhen stratified by age group, children under 2 years (n\u0026thinsp;=\u0026thinsp;66) harbored predominantly non-PCV13 serotypes (84.8%, 56/66). The five most common serotypes were 15B/C (31.0%), followed by 12F/12A/12B/44/46 (12.1%), 38B (10.6%), 8 (11.1%), and 35B (4.5%), together accounting for 66.7% (44/66) of the isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The PCV13 serotypes represented 20% (n\u0026thinsp;=\u0026thinsp;19/95) of all cases, including serotypes 1, 14, 9 V/9A, 23F, and 19F. Among children aged\u0026thinsp;\u0026ge;\u0026thinsp;2 years, the most prevalent serotypes were 15B/C (20.7%, 6/29), 12F/12A/12B/44/46 (20.7%, 6/29), and 1 (20.7%, 6/29), followed by 24F/22A/222B (6.9%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These four types accounted for 69.0% (20/29) of all the serotypes. The less frequent serotypes (3.5% each) included 16F, 38, 8,19F, 9 V/9A and 18C/18F/18B/18A (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMoreover, the rates of vaccine coverage against the serotypes of \u003cem\u003eS. pneumoniae\u003c/em\u003e causing meningitis among children under 5 years of age in Mozambique post-PCV13 introduction in Mozambique (2020\u0026ndash;2023) were 20.0% (19/95), 26.3% (25/95), and 75.8% (72/95) for PCV-13, PCV-15, and PCV-20, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn terms of geographic distribution, serotypes 15B/C, 12F/12A/12B/44/46, 38, 8, and 1 were the most predominant in the northern region of the country (Nampula) after PCV13 introduction, whereas serotypes 15B/C and 12F/12A/12B/44/46 predominated in the central region (Sofala) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In the southern region of the country (Maputo), only one case of serotype 1 and another nontypeable case were recorded.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we reported for the first time the prevalence of non-PCV13 pneumococcal serotypes causing acute bacterial meningitis in children under 5 years four years after the introduction of the PCV13 2p\u0026thinsp;+\u0026thinsp;1 schedule in Mozambique. Our findings revealed a high prevalence of non-PCV13 serotypes (80.0%) compared with the 18.8% reported in our earlier surveillance of confirmed pediatric pneumococcal meningitis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Consequently, only 20% of cases were attributable to PCV13 serotypes, indicating that the current vaccine formulation provides limited coverage in Mozambique. These results are consistent with post-PCV introduction data from other previously reported findings in many African countries [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], Europe [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], Asian countries [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], Latin America [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and North America [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], all of which reported low residual prevalence (~\u0026thinsp;28%) of vaccine-type serotypes following PCV implementation.\u003c/p\u003e\u003cp\u003eIn a 2017 study of \u003cem\u003eS. pneumoniae\u003c/em\u003e isolates from children with acute bacterial meningitis in Mozambique, Nhantumbo et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] reported that PCV13 serotypes 1, 5, 6A, 6B, 23F, 14, 9 V/9A, 4, 3, 19A, and 18A/18B/18C/18F accounted for 81.2% of cases in children under five years of age. Four years later, the present study did not detect several of these serotypes, namely, 5, 3, 6A, 6B, and 19A, highlighting their near elimination. These findings are consistent with previous observations in many African countries, underscoring the impact of immunization strategies in protecting children under 5 years of age from invasive pneumococcal diseases caused by vaccine serotypes [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the present study, the most prevalent non-PCV13 replacement serotypes in Mozambique were 15B/C, 12F/12A/12B/44/468, 22F/22A, 38, 8, 35B, 24F/24A/24B, and 16F. Together, these serotypes accounted for 84.8% of all \u003cem\u003eS. pneumoniae\u003c/em\u003e isolates causing ABM in children aged\u0026thinsp;\u0026lt;\u0026thinsp;2 years. This marked increase in nonvaccine serotypes reflects the serotype replacement phenomenon observed after PCV introduction and aligns with trends reported in other countries following the rollout of pneumococcal conjugate vaccines [\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOverall, serotypes 8, 12F, 15B, 15A, 22F, 33, 38, 8, 35B, 24F/24A/24B, and 16F have emerged as the predominant replacement serotypes recovered from pediatric invasive pneumococcal disease cases worldwide following PCV implementation in both low- and high-income countries [\u003cspan additionalcitationids=\"CR36 CR37 CR38 CR39 CR40 CR41\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This global serotype replacement phenomenon, characterized by the increase in nonvaccine types after the introduction of PCVs, has been repeatedly reported across diverse settings [\u003cspan additionalcitationids=\"CR36 CR37 CR38 CR39 CR40 CR41\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAnother notable finding in our study was that 20.7% of residual serotype 1 cases occurred in children under 2 years of age. However, all of these affected children were unvaccinated (Supplementary Table S2). In many countries, the introduction of PCVs using a 2p\u0026thinsp;+\u0026thinsp;1 schedule has led to a substantial reduction in disease caused by vaccine-type serotypes, including serotype 1 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe geographic distribution of \u003cem\u003eS. pneumomiae serotypes\u003c/em\u003e in Mozambique revealed slight variations. In the northern region of the country (Nampula Province), the predominant serotypes were 15B/C, 12F/12A/12B/44/46, 38, 8, 1, and 14, whereas in the central region of the country (Sofala Province), serotypes 15B/C and 12F/12A/12B/44/46 predominated. In the southern region of the country, only serotype 1 was detected. The frequency and interregional spread of these serotypes can be affected by several factors, such as population mobility, vaccination coverage, public health policies, and environmental conditions [\u003cspan additionalcitationids=\"CR44 CR45 CR46\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRegarding PCV13 serotype coverage, our findings indicate that this vaccine formulation covers only 20.0% (19/95) of the serotypes identified. These results are similar to those reported in several countries in Africa, the Americas, Europe, and Asia, where post-PCV13 coverage rates ranged from 10.0 to 26.4% [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In contrast, higher-valence vaccines, such as PCV20, which includes serotypes 22F, 33F, 8, 10A, 11A, 12F, and 15B/C, cover 75.0% (72/95) of the serotypes identified in our study. These findings are consistent with global data reporting coverage rates of 74.0% for PCV20 [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNampula is one of the most populous provinces in the country, with a total population of 6.102.867 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. A poor resource setting in northern Mozambique, likely related to higher levels of social disadvantage, is associated with significantly higher rates of morbidity across a range of infectious diseases, including pneumococcal disease. Although PCV13 vaccine coverage was greater than 90% nationwide, in Nampula Province, PCV13 vaccine coverage for the 3rd dose decreased from 83.5 to 69.0% in 2020 and 2022, respectively.\u003c/p\u003e\u003cp\u003eIncreased vaccination coverage in the age groups implicated in disease transmission has been reported to generate indirect effects of vaccination programs at the population level [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. With increased indirect effects, the reduction or elimination of transmission and IPD caused by vaccine serotypes can be achieved. Furthermore, the elimination of IPD caused by vaccine serotypes has been reported largely in settings that rapidly achieved very high coverage rates of PCV vaccines [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe acknowledge several limitations that may have influenced our results. First, the data were collected from the three largest hospitals in Mozambique, which may limit the representativeness of our findings. Second, our molecular serotyping method targets only 48 of the most common serotypes/serogroups, which likely explains the few untyped samples, most likely due to the absence of those specific serotypes in our multiplex PCR panel.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study provides, for the first time, evidence of a high prevalence of non-PCV13 serotypes causing pneumococcal meningitis among children under five years of age following the introduction of PCV in Mozambique. Although PCV13 vaccines save many lives in Mozambique, they do not seem to be adequate in addresing some of the highly prevailing non-PCV13 serotypes (15B/C and 12F/12A/12B/44/46). Therefore, we suggest that the country's NITAG reviews available new data and advise that switching to broader-spectrum formulations such as PCV20 may offer better coverage and disease prevention, while the development of a universal pneumococcal vaccine is still pending.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eABM\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eacute bacterial meningitis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eCSF\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecerebrospinal fluid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eCt\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecycle threshold\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eDNA\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edeoxyribonucleic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eBCH\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBeira Central Hospital\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eMCH\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMaputo Central Hospital \u003cb\u003eNCH\u003c/b\u003e:Nampula Central Hospital\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eINS\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInstituto Nacional de Sa\u0026uacute;de\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eIPD\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003einvasive pneumococcal diseases\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eIQR\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003einterquartile range\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eqPCR\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emultiplex qualitative polymerase chain reaction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eNITAG\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNational Immunization Technical Advisory Group\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eNRML\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNational Reference Microbiology Laboratory\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eNTS\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003enontypable specimen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eNVT\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003enonvaccine type\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003ePCV-13\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ethirteen-valent conjugate vaccine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eRRL\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRegional Reference Laboratory\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eOur manuscript does not include any individual personal data; however, consent to participate was obtained from each participant as stated in the Ethics Statement section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eDisclaimer\u003c/h2\u003e\n\u003cp\u003eThe findings and conclusions in this publication are those of the authors and do not necessarily represent the official position of the World Health Organization or the U.S. Centers for Disease Control and Prevention.\u003c/p\u003e\n\u003ch2\u003eInformed Consent\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eStatement\u003c/strong\u003e: Our manuscript does not include any individual personal data; however, consent to participate was obtained from each participant as stated in the Ethics Statement section. The study was performed in accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eGavi, The Vaccine Alliance, reference number: MOZ-HSS-2-INS; WHO Reference: 2014405143-0, creation DFC to support the HIB \u0026amp; Surveillance System.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eE.S.G., I.d.F., A.E.A., A.A.N., C.E.C., Z.C., R.K., E.R.C., L.d.G., E.V.Z., P.I.M., E.F.S., VC., C.D., M.V.N., C.A.F., A.M., A.C.d.F.N.M., and G.W. worked together on study conceptualization, data collection, supervision, and project administration. A.A.N., L.d.G., E.S.G., I.d.F., A.M.M., C.D., and V.C. performed the molecular experiments. E.S.G., R.M., R.K., I.d.F., A.E.A., G.W., and A.A.N. performed the data analysis and validation. A.A.N., I.d.F. and E.S.G. wrote the original draft of the manuscript. I.d.F., E.S.G., A.A.N., C.D., P.I.M., V.C., C.E.C., A.M.M., A.C.F.N.M., A.N.T., G.W., L.d.G., and C.E.C. interpreted the data and wrote the manuscript. All the authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements:\u003c/h2\u003e\n\u003cp\u003eWe thank Marcelino of the Bacteriology Laboratory of the Nampula Central Hospital (NCH), Tim\u0026oacute;tio Bejamim and Joaquim Jo\u0026atilde;o Marcos of the Bacteriology Laboratory of the Beira Central Hospital (BCH), and C\u0026eacute;lio Conjo and Calvina Langa of the Bacteriology Laboratory of the Maputo Central Hospital (MCH) for culturing and identifying the bacterial isolates. We also thank all our colleagues at the National Reference Microbiology Laboratory (NMRL) of the National Institute of Health who received and helped process the samples. We extend our gartitude to CDC-US: Fernanda Lessa, Olivia McGovern, Srinivasan Velusamy, Mahomoudou Ouattara, and Jennifer R. Verani for the country\u0026rsquo;s technical assistance in the implementation and strengthening of the national meningitis surveillance system.\u003c/p\u003e\n\u003ch2\u003eData availability statement:\u003c/h2\u003e\n\u003cp\u003eAll relevant data are presented in the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGBD. 2019. Antimicrobial Resistance Collaborators. Global mortality associated with 33 bacterial pathogens in 2019: A systematic analysis for the Global Burden of Disease Study 2019. Lancet 2022, 400, 2221\u0026ndash;2248. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0140-6736(22)02185-7\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(22)02185-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 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Vaccine. 2017;35:2882\u0026ndash;91. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.vaccine.2017.04.032\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2017.04.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7722758/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7722758/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eIn Mozambique, the introduction of a 13-valent pneumococcal conjugate vaccine (PCV13) using a two plus one schedule began in September 2019. However, the replacement of serotypes with non-PCV types remains a concern worldwide following widespread vaccination. We aim to describe the epidemiology of non-PCV13 types among children under 5 years of age with pneumococcal meningitis four years after PCV13 introduction in Mozambique.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eBetween January 2020 and December 2023, a total of 2,014 cerebrospinal fluid (CSF) samples were collected from eligible children as part of the meningitis surveillance system in Mozambique, 103 (5.1%) of which tested positive for \u003cem\u003eS. pneumoniae\u003c/em\u003e after triplex qPCR. Among these, 86 (83.5%) samples met the quality criteria for serotyping and were subsequently analysed using sequential quadriplex qPCR.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAmong children under 2 years of age, 84.8% of the \u003cem\u003eS. pneumoniae-\u003c/em\u003epositive samples belonged to non-PCV13 serotypes, with serotype 15B/C (31.0%) being the most prevalent, followed by 12F/12A/12B/44/46 (12.1%), 38B (10.6%), and 8 (7.6%). Serotypes 15B/C, 12F/12A/12B/44/46, 38, 8, and 1 were predominant in the northern region of the country (Nampula), whereas serotypes 15B/C and 12F/12A/12B/44/46 were most common in the central region (Sofala). The serotypes coverage of the PCV13, PCV15 and PCV20 vaccine formulations were 20.0%, 26.3% and 75.8%, respectively.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003ePCV13 vaccines save many lives in Mozambique. However, they do not seem to be adequate in addresing some of the highly prevailing non-PCV13 serotypes (15B/C and 12F/12A/12B/44/46). Therefore, we suggest that the country's National Immunization Technical Advisory Group (NITAG) review new data and advice on switching to extended valency vaccines such as PCV20.\u003c/p\u003e","manuscriptTitle":"Emergence of nonvaccine Streptococcus pneumoniae following a 13-valent pneumococcal conjugate vaccine and schedule change in Mozambique: implications for a national immunization strategy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-04 01:20:28","doi":"10.21203/rs.3.rs-7722758/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-30T08:32:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-28T17:15:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-23T00:27:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287170316856500386077322468124577718328","date":"2025-12-18T11:21:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167711352835972952195191183779088971415","date":"2025-12-17T06:54:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22981588084161145485257509399494251893","date":"2025-12-16T07:29:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"116168967027035598651460551326016839283","date":"2025-12-14T15:53:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"269496618220043900953040451154587462261","date":"2025-12-07T10:34:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-02T00:55:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-29T12:09:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24911770205638133074219394324101086102","date":"2025-11-28T06:08:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-13T06:41:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298956935948302267967435770160109248479","date":"2025-11-12T10:04:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212628418904738974864597225061952316735","date":"2025-11-10T12:11:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312303622561163851750400174480005447816","date":"2025-11-10T00:26:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13915219322933890524474965969648039815","date":"2025-11-08T10:11:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-03T22:29:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69482115444968803606052132195956759876","date":"2025-10-22T16:40:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334116930893029232133024662957409932020","date":"2025-10-22T14:33:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-22T13:50:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-03T09:17:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-01T10:58:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-01T10:57:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2025-09-26T14:20:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"784882a2-e7af-4f8c-9726-09633cf9bb2d","owner":[],"postedDate":"November 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2025-12-30T08:39:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-04 01:20:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7722758","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7722758","identity":"rs-7722758","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}