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•We present updated recommendations from a World Health Organization working group.•These are a core set of standard methods for pneumococcal carriage studies.•Methods for the collection, transport and storage of nasopharyngeal samples are outlined.•Methods for identification and serotyping of pneumococci are described.•The epidemiological rationale for pneumococcal carriage studies is described. In 2003 the World Health Organization (WHO) convened a working group and published a set of standard methods for studies measuring nasopharyngeal carriage of Streptococcus pneumoniae (the pneumococcus). The working group recently reconvened under the auspices of the WHO and updated the consensus standard methods. These methods describe the collection, transport and storage of nasopharyngeal samples, as well as provide recommendations for the identification and serotyping of pneumococci using culture and non-culture based approaches. We outline the consensus position of the working group, the evidence supporting this position, areas worthy of future research, and the epidemiological role of carriage studies. Adherence to these methods will reduce variability in the conduct of pneumococcal carriage studies undertaken in the context of pneumococcal vaccine trials, implementation studies, and epidemiology studies more generally so variability in methodology does not confound the interpretation of study findings.

Streptococcus pneumoniae is an important cause of infection and commonly colonizes the nasopharynx of young children, along with other potentially pathogenic bacteria. The objectives of this study were to estimate the carriage prevalence of S. pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus in young children in Indonesia, and to examine interactions between these bacterial species. 302 healthy children aged 12-24 months were enrolled in community health centers in the Bandung, Central Lombok, and Padang regions. Nasopharyngeal swabs were collected and stored according to World Health Organization recommendations, and bacterial species detected by qPCR. Pneumococcal serotyping was conducted by microarray and latex agglutination/Quellung. Overall carriage prevalence was 49.5% for S. pneumoniae, 27.5% for H. influenzae, 42.7% for M. catarrhalis, and 7.3% for S. aureus. Prevalence of M. catarrhalis and S. pneumoniae, as well as pneumococcal serotype distribution, varied by region. Positive associations were observed for S. pneumoniae and M. catarrhalis (OR 3.07 [95%CI 1.91-4.94]), and H. influenzae and M. catarrhalis (OR 2.34 [95%CI 1.40-3.91]), and a negative association was found between M. catarrhalis and S. aureus (OR 0.06 [95%CI 0.01-0.43]). Densities of S. pneumoniae, H. influenzae, and M. catarrhalis were positively correlated when two of these species were present. Prior to pneumococcal vaccine introduction, pneumococcal carriage prevalence and serotype distribution varies among children living in different regions of Indonesia. Positive associations in both carriage and density identified among S. pneumoniae, H. influenzae, and M. catarrhalis suggest a synergistic relationship among these species with potential clinical implications.

Limited data from Asia are available on long-term effects of pneumococcal conjugate vaccine introduction on pneumococcal carriage. Here we assess the impact of 13-valent pneumococcal conjugate vaccine (PCV13) introduction on nasopharyngeal pneumococcal carriage prevalence, density and antimicrobial resistance. Cross-sectional carriage surveys were conducted pre-PCV13 (2015) and post-PCV13 introduction (2017 and 2022). Pneumococci were detected and quantified by real-time PCR from nasopharyngeal swabs. DNA microarray was used for molecular serotyping and to infer genetic lineage (Global Pneumococcal Sequence Cluster). The study included 1461 infants (5–8 weeks old) and 1489 toddlers (12–23 months old) enrolled from family health clinics. We show a reduction in PCV13 serotype carriage (with non-PCV13 serotype replacement) and a reduction in the proportion of samples containing resistance genes in toddlers six years post-PCV13 introduction. We observed an increase in pneumococcal nasopharyngeal density. Serotype 15 A, the most prevalent non-vaccine-serotype in 2022, was comprised predominantly of GPSC904;9. Reductions in PCV13 serotype carriage will likely result in pneumococcal disease reduction. It is important for ongoing surveillance to monitor serotype changes to potentially inform new vaccine development. Pneumococcal carriage is a useful outcome to evaluate pneumococcal conjugate vaccine (PCV) introduction. Here the authors conducted cross-sectional carriage surveys before (2015) and after 13-valent PCV introduction (2017 and 2022) in Mongolia to determine vaccine impact.

Starting in June 2016, the 13-valent pneumococcal conjugate vaccine (PCV13) was introduced into the routine immunization program of Mongolia by using a 2+1 dosing schedule, phased by district. We used prospective hospital surveillance to evaluate the vaccine's effect on pneumonia incidence rates among children 2-59 months of age over a 6-year period. Of 17,607 children with pneumonia, overall adjusted incidence rate ratios showed decreased primary endpoint pneumonia, very severe pneumonia, and probable pneumococcal pneumonia until June 2021. Results excluding and including the COVID-19 pandemic period were similar. Pneumonia declined in 3 districts that introduced PCV13 with catch-up campaigns but not in the 1 district that did not. After PCV13 introduction, vaccine-type pneumococcal carriage prevalence decreased by 44% and nonvaccine-type carriage increased by 49%. After PCV13 introduction in Mongolia, the incidence of more specific pneumonia endpoints declined in children 2-59 months of age; additional benefits were conferred by catch-up campaigns.

The maternal pregnancy microbiome (including genitourinary and gut) has been linked to important pregnancy/birth and later childhood health outcomes. However, such sampling as part of large population cohort studies is logistically and financially challenging. Many countries routinely collect vaginal or vaginal-rectal swabs in late pregnancy for Group B Streptococcus (GBS) screening, but their utility for population-based research is still unclear. As part of planning for the Generation Victoria population-based cohort study beginning in pregnancy, we assessed the utility and reliability of residual clinical GBS vaginal/vaginal-rectal swabs for generating late pregnancy microbiome data. We carried out a two-phased pilot study. Phase one assessed the level of microbial diversity apparent in ‘residual’ clinical vaginal/vaginal-rectal swabs post clinical testing and storage for 7–10 days at 4 °C (routine clinical practice). Phase two directly assessed the impact of storage time and temperature on the microbial composition of vaginal/vaginal-rectal swabs collected specifically for research purposes. The microbiota composition in the ‘residual’ clinical swabs aligned with published studies. The ‘research’ swabs, stored at 4 °C for up to ten days, showed minimal changes in microbiota profile, compared to swabs examined on the day of collection. In contrast, significant variation in diversity was seen in swabs stored at room temperature for up to 48 h. Residual clinical material from swabs collected primarily for GBS screening in late pregnancy represent a reliable and abundant source of material for assessing the late pregnancy maternal microbiome for research purposes. This represents a low-burden opportunity for population-representative pregnancy studies to assess the potential of late pregnancy microbiome for prediction and understanding maternal and child health outcomes.

The pneumococcus is a diverse pathogen whose primary niche is the nasopharynx. Over 90 different serotypes exist, and nasopharyngeal carriage of multiple serotypes is common. Understanding pneumococcal carriage is essential for evaluating the impact of pneumococcal vaccines. Traditional serotyping methods are cumbersome and insufficient for detecting multiple serotype carriage, and there are few data comparing the new methods that have been developed over the past decade. We established the PneuCarriage project, a large, international multi-centre study dedicated to the identification of the best pneumococcal serotyping methods for carriage studies. Reference sample sets were distributed to 15 research groups for blinded testing. Twenty pneumococcal serotyping methods were used to test 81 laboratory-prepared (spiked) samples. The five top-performing methods were used to test 260 nasopharyngeal (field) samples collected from children in six high-burden countries. Sensitivity and positive predictive value (PPV) were determined for the test methods and the reference method (traditional serotyping of >100 colonies from each sample). For the alternate serotyping methods, the overall sensitivity ranged from 1% to 99% (reference method 98%), and PPV from 8% to 100% (reference method 100%), when testing the spiked samples. Fifteen methods had ≥70% sensitivity to detect the dominant (major) serotype, whilst only eight methods had ≥70% sensitivity to detect minor serotypes. For the field samples, the overall sensitivity ranged from 74.2% to 95.8% (reference method 93.8%), and PPV from 82.2% to 96.4% (reference method 99.6%). The microarray had the highest sensitivity (95.8%) and high PPV (93.7%). The major limitation of this study is that not all of the available alternative serotyping methods were included. Most methods were able to detect the dominant serotype in a sample, but many performed poorly in detecting the minor serotype populations. Microarray with a culture amplification step was the top-performing method. Results from this comprehensive evaluation will inform future vaccine evaluation and impact studies, particularly in low-income settings, where pneumococcal disease burden remains high.

Pleural empyema is a serious complication of pneumonia in children. Negative bacterial cultures commonly impede optimal antibiotic therapy. To improve bacterial identification, we developed a molecular assay and evaluated its performance compared with bacterial culture. Our multiplex-quantitative PCR to detect Streptococcus pneumoniae , Streptococcus pyogenes , Staphylococcus aureus and Haemophilus influenzae was assessed using bacterial genomic DNA and laboratory-prepared samples (n = 267). To evaluate clinical performance, we conducted the Molecular Assessment of Thoracic Empyema (MATE) observational study, enrolling children hospitalised with empyema. Pleural fluids were tested by bacterial culture and multiplex-qPCR, and performance determined using a study gold standard. We determined clinical sensitivity and time-to-organism-identification to assess the potential of the multiplex-qPCR to reduce the duration of empiric untargeted antibiotic therapy. Using spiked samples, the multiplex-qPCR demonstrated 213/215 (99.1%) sensitivity and 52/52 (100%) specificity for all organisms. During May 2019–March 2023, 100 children were enrolled in the MATE study; median age was 3.9 years (IQR 2–5.6). A bacterial pathogen was identified in 90/100 (90%) specimens by multiplex-qPCR, and 24/100 (24%) by bacterial culture ( P <0.001). Multiplex-qPCR identified a bacterial cause in 68/76 (90%) culture-negative specimens. S . pneumoniae was the most common pathogen, identified in 67/100 (67%) specimens. We estimate our multiplex-qPCR would have reduced the duration of untargeted antibiotic therapy in 61% of cases by a median 20 days (IQR 17.5–23, range 1–55). Multiplex-qPCR significantly increased pathogen detection compared with culture and may allow for reducing the duration of untargeted antibiotic therapy.

Nasopharyngeal carriage of Streptococcus pneumoniae (the pneumococcus) is a precursor to pneumococcal disease. Several host and environmental factors have been associated with pneumococcal carriage, however few studies have examined the relationship between host factors and pneumococcal carriage density. We sought to identify risk factors for pneumococcal carriage and density using data from cross-sectional pneumococcal carriage surveys conducted in the Lao People's Democratic Republic before and after the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13). Nasopharyngeal swabs were collected infants from aged 5-8 weeks old (n = 999) and children aged 12-23 months (n = 1,010), pneumococci detected by quantitative PCR, and a risk factor questionnaire completed. Logistic and linear regression models were used to evaluate associations between participant characteristics and pneumococcal carriage and density. In infants aged 5-8 weeks, living in a household with two or more children under the age of five years (aOR 1.97; 95% CI 1.39-2.79) and low family income (aOR 1.64; 95% CI 0.99-2.72) were positively associated with pneumococcal carriage. For children aged 12-23 months, upper respiratory tract infection (URTI) symptoms (aOR 2.64; 95% CI 1.97-3.53), two or more children under five in the household (aOR 2.40; 95% CI 1.80-3.20), and rural residence (aOR 1.84, 95% CI 1.35-2.50) were positively associated with pneumococcal carriage. PCV13 vaccination was negatively associated with carriage of PCV13 serotypes (aOR 0.60; 95% CI 0.44-0.83). URTI symptoms (p < 0.001), current breastfeeding (p = 0.005), rural residence (p = 0.012), and delivery by Caesarean section (p = 0.035) were associated with higher mean pneumococcal density in pneumococcal carriers (both age groups combined). This study provides new data on pneumococcal carriage and density in a high disease burden setting in southeast Asia.

There is growing evidence on the importance of bacterial/viral interaction in the course of pneumonia. In Laos, no study has investigated respiratory pathogen co-detection. We conducted a study at Mahosot Hospital in Vientiane to determine whether bacterial/viral co-detection and pneumococcal density are associated with severe pneumonia. Between December 2013 and December 2016, 934 under 5 years old hospitalized children with ARI were enrolled. Swabs from the upper respiratory tract were collected and analyzed by real-time PCR. The most common co-detected microorganisms were Streptococcus pneumoniae / Haemophilus influenzae (24%), Respiratory Syncytial Virus (RSV)/ S. pneumoniae (12%) and RSV/ H. influenzae (16%). Pneumococcal density was 4.52 times higher in influenza virus positive participants. RSV/ S. pneumoniae and RSV/ H. influenzae co-detections were positively associated with severe pneumonia in univariate analysis (OR 1.86, 95%CI:1.22–2.81, p  = 0.003 and OR 2.09, 95%CI:1.46-3.00), but not confirmed in adjusted analysis (aOR 0.72, 95%CI:0.38–1.6, p  = 0.309 and aOR 1.37, 95%CI:0.73–2.58). In RSV positive patients, there was no association between pneumococcal density and severe pneumonia. Our findings confirmed an association between pneumococcal density and influenza but not RSV severe pneumonia in young children. Results highlight the complexity of the interaction of viral/bacterial pathogens, which might not have a simple synergistic action in the evolution of pneumonia.

Respiratory bacterial-viral coinfections (such as pneumococci and influenza virus) are often synergistic, resulting in enhanced disease severity. Although colonization of the nasopharynx is the precursor to disease and transmission, little is known about bacterial-viral interactions that occur within this niche. Streptococcus pneumoniae (the pneumococcus) is a leading cause of pneumonia in children under 5 years of age. Coinfection by pneumococci and respiratory viruses enhances disease severity. Little is known about pneumococcal coinfections with respiratory syncytial virus (RSV). Here, we developed a novel infant mouse model of coinfection using pneumonia virus of mice (PVM), a murine analogue of RSV, to examine the dynamics of coinfection in the upper respiratory tract, an anatomical niche that is essential for host-to-host transmission and progression to disease. Coinfection increased damage to the nasal tissue and increased production of the chemokine CCL3. Nasopharyngeal pneumococcal density and shedding in nasal secretions were increased by coinfection. In contrast, coinfection reduced PVM loads in the nasopharynx, an effect that was independent of pneumococcal strain and the order of infection. We showed that this “antagonistic” effect was absent using either ethanol-killed pneumococci or a pneumococcal mutant deficient in capsule production and incapable of nasopharyngeal carriage. Colonization with a pneumococcal strain naturally unable to produce capsule also reduced viral loads. The pneumococcus-mediated reduction in PVM loads was caused by accelerated viral clearance from the nasopharynx. Although these synergistic and antagonistic effects occurred with both wild-type pneumococcal strains used in this study, the magnitude of the effects was strain dependent. Lastly, we showed that pneumococci can also antagonize influenza virus. Taken together, our study has uncovered multiple novel facets of bacterial-viral coinfection. Our findings have important public health implications, including for bacterial and viral vaccination strategies in young children. IMPORTANCE Respiratory bacterial-viral coinfections (such as pneumococci and influenza virus) are often synergistic, resulting in enhanced disease severity. Although colonization of the nasopharynx is the precursor to disease and transmission, little is known about bacterial-viral interactions that occur within this niche. In this study, we developed a novel mouse model to examine pneumococcal-viral interactions in the nasopharynx with pneumonia virus of mice (PVM) and influenza. We found that PVM infection benefits pneumococci by increasing their numbers in the nasopharynx and shedding of these bacteria in respiratory secretions. In contrast, we discovered that pneumococci decrease PVM numbers by accelerating viral clearance. We also report a similar effect of pneumococci on influenza. By showing that coinfections lead to both synergistic and antagonistic outcomes, our findings challenge the existing dogma in the field. Our work has important applications and implications for bacterial and viral vaccines that target these microbes.