Highlights • 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.
Formyl peptide receptor 2/lipoxin A
(LXA
) receptor (Fpr2/ALX) co-ordinates the transition from inflammation to resolution during acute infection by binding to distinct ligands including serum ...amyloid A (SAA) and Resolvin D1 (RvD1). Here, we evaluated the proresolving actions of aspirin-triggered RvD1 (AT-RvD1) in an acute coinfection pneumonia model. Coinfection with
and influenza A virus (IAV) markedly increased pneumococcal lung load and neutrophilic inflammation during the resolution phase. Fpr2/ALX transcript levels were increased in the lungs of coinfected mice, and immunohistochemistry identified prominent Fpr2/ALX immunoreactivity in bronchial epithelial cells and macrophages. Levels of circulating and lung SAA were also highly increased in coinfected mice. Therapeutic treatment with exogenous AT-RvD1 during the acute phase of infection (day 4-6 post-pneumococcal inoculation) significantly reduced the pneumococcal load. AT-RvD1 also significantly reduced neutrophil elastase (NE) activity and restored total antimicrobial activity in bronchoalveolar lavage (BAL) fluid (BALF) of coinfected mice. Pneumonia severity, as measured by quantitating parenchymal inflammation or alveolitis was significantly reduced with AT-RvD1 treatment, which also reduced the number of infiltrating lung neutrophils and monocytes/macrophages as assessed by flow cytometry. The reduction in distal lung inflammation in AT-RvD1-treated mice was not associated with a significant reduction in inflammatory and chemokine mediators. In summary, we demonstrate that in the coinfection setting, SAA levels were persistently increased and exogenous AT-RvD1 facilitated more rapid clearance of pneumococci in the lungs, while concurrently reducing the severity of pneumonia by limiting excessive leukocyte chemotaxis from the infected bronchioles to distal areas of the lungs.
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.
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.
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.
A number of pneumococcal carriage studies in children have been conducted in recent years. However, summary data of carriage prevalence and serotype distribution from South East Asia Region (SEAR) ...are limited. This may lead to the misconception that Streptococcus pneumoniae vaccine-types are uncommon in the region. Systematic reviews of pneumococcal carriage and the distribution of serotypes are critically important for evidence-based decision-making. We aimed to summarize published data on the serotype prevalence of S. pneumoniae carried in the nasopharynx of children under 5 years of age in SEAR. We performed a systematic review and meta-analysis for relevant studies on S. pneumoniae carriage conducted prior to PCV program implementation from online journal databases published between January 2001 to December 2019. The pooled prevalence of S. pneumoniae in healthy children under 5 years of age in SEAR was 36.0% (95% CI 34.2%–37.8%), and ranged from 68.0% (95% CI: 61.9%–74.0%) in Cambodia to 7.6% (95% CI: 5.7%–9.6%) in Malaysia. Serotypes 6A/B, 23F and 19F were the most common serotypes in children <5 years, accounting for 12.9% (95% CI: 9.4%–16.3%), 9.3% (95% CI: 5.9%–12.8%) and 10.1% (95% CI: 6.6%–13.5%) of isolates, respectively. Vaccine policy makers should take these results into account when making decisions on pneumococcal conjugate vaccine programs implementation. Given the paucity of data, collection of more extensive and updated information of S. pneumoniae serotype epidemiology in children under five years in SEAR is also very important for future studies.
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.
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.
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.
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.