Insights Into Pneumococcal Pneumonia Using Lung Aspirates and Nasopharyngeal Swabs Collected From Pneumonia Patients in The Gambia.

BACKGROUND: We investigated the pathogenesis of pneumococcal pneumonia using clinical specimens collected for pneumonia surveillance in The Gambia. METHODS: Lung aspirates and nasopharyngeal swabs from 31 patients were examined by culture, quantitative polymerase chain reaction (qPCR), whole genome sequencing, serotyping, and reverse-transcription qPCR. RESULTS: Five lung aspirates cultured pneumococci, with a matching strain identified in the nasopharynx. Three virulence genes including ply (pneumolysin) were upregulated >20-fold in the lung compared with the nasopharynx. Nasopharyngeal pneumococcal density was higher in pediatric pneumonia patients compared with controls (P < .0001). CONCLUSIONS: Findings suggest that changes in pneumococcal gene expression occurring in the lung environment may be important in pathogenesis.

Streptococcus pneumoniae serotype 33G: genetic, serological, and structural analysis of a new capsule type.

IMPORTANCE: Streptococcus pneumoniae (the pneumococcus) is a bacterial pathogen with the greatest burden of disease in Asia and Africa. The pneumococcal capsular polysaccharide has biological relevance as a major virulence factor as well as public health importance as it is the target for currently licensed vaccines. These vaccines have limited valency, covering up to 23 of the >100 known capsular types (serotypes) with higher valency vaccines in development. Here, we have characterized a new pneumococcal serotype, which we have named 33G. We detected serotype 33G in nasopharyngeal swabs (n = 20) from children and adults hospitalized with pneumonia, as well as healthy children in Mongolia. We show that the genetic, serological, and biochemical properties of 33G differ from existing serotypes, satisfying the criteria to be designated as a new serotype. Future studies should focus on the geographical distribution of 33G and any changes in prevalence following vaccine introduction.

Variants of Streptococcus pneumoniae Serotype 14 from Papua New Guinea with the Potential to Be Mistyped and Escape Vaccine-Induced Protection.

Streptococcus pneumoniae (the pneumococcus) is a human pathogen of global importance, classified into serotypes based on the type of capsular polysaccharide produced. Serotyping of pneumococci is essential for disease surveillance and vaccine impact measurement. However, the accuracy of serotyping methods can be affected by previously undiscovered variants. Previous studies have identified variants of serotype 14, a highly invasive serotype included in all licensed vaccine formulations. However, the potential of these variants to influence serotyping accuracy and evade vaccine-induced protection has not been investigated. In this study, we screened 1,386 nasopharyngeal swabs from children hospitalized with acute respiratory infection in Papua New Guinea for pneumococci. Swabs containing pneumococci (n = 1,226) were serotyped by microarray to identify pneumococci with a divergent serotype 14 capsule locus. Three serotype 14 variants ('14-like') were isolated and characterized further. The serotyping results of these isolates using molecular methods varied depending on the method, with 3/3 typing as nontypeable (PneumoCaT), 3/3 typing as serotype 14 (seroBA), and 2/3 typing as serotype 14 (SeroCall and quantitative PCR). All three isolates were nontypeable by phenotypic methods (Quellung and latex agglutination), indicating the absence of capsule. Illumina and nanopore sequencing were employed to examine their capsule loci and revealed unique mutations. Lastly, when incubated with sera from vaccinated individuals, the 14-like isolates evaded serotype-specific opsonophagocytic killing. Our study highlights the need for phenotypic testing to validate serotyping data derived from molecular methods. The convergent evolution of capsule loss underscores the importance of studying pneumococcal population biology to monitor the emergence of pneumococci capable of vaccine escape, globally. IMPORTANCE Pneumococcus is a pathogen of major public health importance. Current vaccines have limited valency, targeting a subset (up to 20) of the more than 100 capsule types (serotypes). Precise serotyping methods are therefore essential to avoid mistyping, which can reduce the accuracy of data used to inform decisions around vaccine introduction and/or maintenance of national vaccination programs. In this study, we examine a variant of serotype 14 (14-like), a virulent serotype present in all currently licensed vaccine formulations. Although these 14-like pneumococci no longer produce a serotype 14 capsule, widely used molecular methods can mistype them as serotype 14. Importantly, we show that 14-like pneumococci can evade opsonophagocytic killing mediated by vaccination. Despite the high accuracy of molecular methods for serotyping, our study reemphasizes their limitations. This is particularly relevant in situations where nonvaccine type pneumococci (e.g., the 14-likes in this study) could potentially be misidentified as a vaccine type (e.g., serotype 14).

Evaluation strategies for measuring pneumococcal conjugate vaccine impact in low-resource settings.

OBJECTIVES: Pneumococcal conjugate vaccines (PCVs) are effective in reducing pneumococcal disease. We measured 13-valent PCV (PCV13) effect on different pneumococcal outcomes using diverse studies in Lao People's Democratic Republic. METHODS: Studies included: pre-PCV13 population-based record review of hospitalized childhood pneumonia cases; acute respiratory infection (ARI) study post-PCV13 to demonstrate effectiveness (VE) against hypoxic pneumonia; invasive pneumococcal disease (IPD) surveillance in all ages (2004-2018); carriage studies in children hospitalized with ARI (2013-2019); community carriage surveys pre- and post-PCV13. RESULTS: Annual pneumonia incidence rate in children pre-PCV13 was 1,530 (95% confidence interval [CI] 1,477-1,584) per 100,000. Adjusted VE against hypoxic pneumonia was 37% (95% CI 6-57%). For IPD, 85% (11/13) of cases were due to vaccine-types pre-PCV13, and 43% (3/7) post-PCV13 in children aged <5 years; for ≥5 years, 61% (27/44) and 42% (17/40), respectively. For ARI cases, adjusted VE for vaccine-type carriage was 39% (95% CI 4-60) in <5 year olds; slightly higher than community surveys (23% [95% CI 4-39%] in 12-23 month olds). CONCLUSIONS: Despite limited baseline data, we found evidence of PCV13 impact on disease and carriage. Our approach could be used in similar settings to augment existing WHO PCV evaluation guidelines.

IMMUNOGENICITY AND IMPACT ON NASOPHARYNGEAL CARRIAGE OF A SINGLE DOSE OF PCV10 GIVEN TO VIETNAMESE CHILDREN AT 18 MONTHS OF AGE.

BACKGROUND: This study investigated the immunogenicity and impact on nasopharyngeal carriage of a single dose of PCV10 given to 18-month-old Vietnamese children. This information is important for countries considering catch-up vaccination during PCV introduction and in the context of vaccination during humanitarian crises. METHODS: Two groups of PCV-naïve children within the Vietnam Pneumococcal Project received PCV10 (n=197) or no PCV (unvaccinated; n=199) at 18 months of age. Blood samples were collected at 18, 19, and 24 months of age, and nasopharyngeal swabs at 18 and 24 months of age. Immunogenicity was assessed by measuring serotype-specific IgG, opsonophagocytosis (OPA) and memory B cells (Bmem). Pneumococci were detected and quantified using real-time PCR and serotyped by microarray. FINDINGS: At 19 months of age, IgG and OPA responses were higher in the PCV10 group compared with the unvaccinated group for all PCV10 serotypes and cross-reactive serotypes 6A and 19A. This was sustained out to 24 months of age, at which point PCV10-type carriage was 60% lower in the PCV10 group than the unvaccinated group. Bmem levels increased between 18 and 24 months of age in the vaccinated group. INTERPRETATION: We demonstrate strong protective immune responses in vaccinees following a single dose of PCV10 at 18 months of age, and a potential impact on herd protection through a substantial reduction in vaccine-type carriage. A single dose of PCV10 in the second year of life could be considered as part of catch-up campaigns or in humanitarian crises to protect children at high-risk of pneumococcal disease.

Molecular epidemiology of Streptococcus pneumoniae serogroup 6 isolates from Fijian children, including newly identified serotypes 6C and 6D.

Multilocus sequence typing (MLST) was applied to all unique serotype 6C and 6D isolates and a random selection of serotype 6B and 6A isolates from nasopharyngeal swabs from Fijian children enrolled in a recent vaccine trial. The results suggest that Fijian serotype 6D has arisen independently from both serotypes 6A/C and 6B.

Factors associated with pneumococcal carriage and density in children and adults in Fiji, using four cross-sectional surveys.

This study describes predictors of pneumococcal nasopharyngeal carriage and density in Fiji. We used data from four annual (2012-2015) cross-sectional surveys, pre- and post-introduction of ten-valent pneumococcal conjugate vaccine (PCV10) in October 2012. Infants (5-8 weeks), toddlers (12-23 months), children (2-6 years), and their caregivers participated. Pneumococci were detected and quantified using lytA qPCR, with molecular serotyping by microarray. Logistic and quantile regression were used to determine predictors of pneumococcal carriage and density, respectively. There were 8,109 participants. Pneumococcal carriage was negatively associated with years post-PCV10 introduction (global P<0.001), and positively associated with indigenous iTaukei ethnicity (aOR 2.74 [95% CI 2.17-3.45] P<0.001); young age (infant, toddler, and child compared with caregiver participant groups) (global P<0.001); urban residence (aOR 1.45 [95% CI 1.30-2.57] P<0.001); living with ≥2 children <5 years of age (aOR 1.42 [95% CI 1.27-1.59] P<0.001); low family income (aOR 1.44 [95% CI 1.28-1.62] P<0.001); and upper respiratory tract infection (URTI) symptoms (aOR 1.77 [95% CI 1.57-2.01] P<0.001). Predictors were similar for PCV10 and non-PCV10 carriage, except PCV10 carriage was negatively associated with PCV10 vaccination (0.58 [95% CI 0.41-0.82] P = 0.002) and positively associated with exposure to household cigarette smoke (aOR 1.21 [95% CI 1.02-1.43] P = 0.031), while there was no association between years post-PCV10 introduction and non-PCV10 carriage. Pneumococcal density was positively associated with URTI symptoms (adjusted median difference 0.28 [95% CI 0.16, 0.40] P<0.001) and toddler and child, compared with caregiver, participant groups (global P = 0.008). Predictors were similar for PCV10 and non-PCV10 density, except infant, toddler, and child participant groups were not associated with PCV10 density. PCV10 introduction was associated with reduced the odds of overall and PCV10 pneumococcal carriage in Fiji. However, after adjustment iTaukei ethnicity was positively associated with pneumococcal carriage compared with Fijians of Indian Descent, despite similar PCV10 coverage rates.

Associations between ethnicity, social contact, and pneumococcal carriage three years post-PCV10 in Fiji.

BACKGROUND: Pneumococcal carriage is a prerequisite for pneumococcal disease. Little is known about whether social contact frequency and intensity are associated with pneumococcal carriage. In Fiji, indigenous iTaukei have higher prevalence of pneumococcal carriage compared with Fijians of Indian Descent (FID). We hypothesised that contact differences may contribute to ethnic differences in pneumococcal carriage prevalence and density. METHODS: In 2015, young infants (5-8 weeks), toddlers (12-23 months), children (2-6 years), and caregivers from Suva and surrounding areas, participated in a cross-sectional survey (n = 2014), three years post pneumococcal conjugate vaccine introduction. Demographic and contact data, and nasopharyngeal swabs were collected. Pneumococci were detected, and quantified using quantitative real-time PCR, with molecular serotyping by microarray. Associations between ethnicity, contact, and pneumococcal carriage and density were estimated using multivariable generalised estimating equation regression models. RESULTS: iTaukei participants had larger household sizes, higher pneumococcal carriage rates, more contacts, and more frequent contacts of longer duration, compared with FID. The odds of vaccine-type carriage increased by 28% (95% CI 8-53%) P < 0.01 in association with physical contact with 7-14 year old children. iTaukei ethnicity was associated with vaccine-type carriage (aOR) 1.73; 95% CI 1.06-2.82, P = 0.03) and non-vaccine type carriage (aOR 5.98; 95% CI 4.47-8.00, P < 0.01). Ethnicity and contact were not associated with pneumococcal density. CONCLUSIONS: iTaukei had greater frequency and intensity of contact compared with FID. Physical contact was associated with pneumococcal carriage. Observed differences in pneumococcal nasopharyngeal carriage prevalence between iTaukei and FID were not explained by differences in social contact patterns by ethnicity.

Pneumococcal carriage in children in Ulaanbaatar, Mongolia before and one year after the introduction of the 13-valent pneumococcal conjugate vaccine.

BACKGROUND: Nasopharyngeal carriage of Streptococcus pneumoniae precedes disease, is the source of pneumococcal community spread, and the mechanism for herd protection provided by pneumococcal conjugate vaccines (PCVs). There are few PCV impact studies in low- and middle-income countries, particularly in Asia. In 2016, Mongolia introduced the 13-valent PCV (PCV13) in a phased manner using a 2 + 1 schedule, with catch-up. We aimed to assess the impact of PCV13 introduction on nasopharyngeal pneumococcal carriage and density in children in Mongolia. METHODS: We conducted two cross-sectional carriage surveys (pre- and one year post-PCV) at community health clinics in two districts of the capital city, Ulaanbaatar in both May-July 2015 and 2017. The study analysis included 961 children too young to be vaccinated (5-8 weeks old) and 989 children eligible for vaccination (12-23 months old). Pneumococci were detected by quantitative real-time PCR and molecular serotyping performed using DNA microarray. FINDINGS: One year post-PCV introduction, PCV13 serotype carriage reduced by 52% in 12-23 month olds (adjusted prevalence ratio [aPR] 0.48 [95% confidence interval [CI] 0.39-0.59]), with evidence of non-PCV13 serotype replacement (aPR 1.55 [95% CI 1.30-1.85]), compared with the pre-PCV period. In 5-8 week olds, PCV13 serotype carriage reduced by 51% (aPR 0.49 [95% CI 0.33-0.73]) with no significant change in non-PCV13 serotype carriage (aPR 1.10 [95% CI 0.83-1.46]). An increase was observed in both PCV13 and non-PCV13 pneumococcal density post-PCV introduction. Antimicrobial resistance (AMR) genes were common, with 82.3% of samples containing at least one of the 10 AMR genes assessed. CONCLUSION: This study demonstrates substantive PCV13 impact on pneumococcal carriage one year post-vaccine introduction in Mongolia. The reductions in PCV13 serotype carriage are likely to result in reductions in pneumococcal disease including indirect effects. Increases in non-PCV13 serotypes require further monitoring.

Pneumococcal carriage in vaccine-eligible children and unvaccinated infants in Lao PDR two years following the introduction of the 13-valent pneumococcal conjugate vaccine.

Pneumococcal carriage is a prerequisite for disease, and underpins herd protection provided by pneumococcal conjugate vaccines (PCVs). There are few data on the impact of PCVs in lower income settings, particularly in Asia. In 2013, the Lao People's Democratic Republic (Lao PDR) introduced 13-valent PCV (PCV13) as a 3 + 0 schedule (doses at 6, 10 and 14 weeks of age) with limited catch-up vaccination. We conducted two cross-sectional carriage surveys (pre- and two years post-PCV) to assess the impact of PCV13 on nasopharyngeal pneumococcal carriage in 5-8 week old infants (n = 1000) and 12-23 month old children (n = 1010). Pneumococci were detected by quantitative real-time PCR, and molecular serotyping was performed using DNA microarray. Post PCV13, there was a 23% relative reduction in PCV13-type carriage in children aged 12-23 months (adjusted prevalence ratio [aPR] 0.77 [0.61-0.96]), and no significant change in non-PCV13 serotype carriage (aPR 1.11 [0.89-1.38]). In infants too young to be vaccinated, there was no significant change in carriage of PCV13 serotypes (aPR 0.74 [0.43-1.27]) or non-PCV13 serotypes (aPR 1.29 [0.85-1.96]), although trends were suggestive of indirect effects. Over 70% of pneumococcal-positive samples contained at least one antimicrobial resistance gene, which were more common in PCV13 serotypes (p < 0.001). In 12-23 month old children, pneumococcal density of both PCV13 serotypes and non-PCV13 serotypes was higher in PCV13-vaccinated compared with undervaccinated children (p = 0.004 and p < 0.001, respectively). This study provides evidence of PCV13 impact on carriage in a population without prior PCV7 utilisation, and provides important data from a lower-middle income setting in Asia. The reductions in PCV13 serotype carriage in vaccine-eligible children are likely to result in reductions in pneumococcal transmission and disease in Lao PDR.

Discovery of a Streptococcus pneumoniae serotype 33F capsular polysaccharide locus that lacks wcjE and contains a wcyO pseudogene.

As part of large on-going vaccine impact studies in Fiji and Mongolia, we identified 25/2750 (0.9%) of nasopharyngeal swabs by microarray that were positive for Streptococcus pneumoniae contained pneumococci with a divergent 33F capsular polysaccharide locus (designated '33F-1'). We investigated the 33F-1 capsular polysaccharide locus to better understand the genetic variation and its potential impact on serotyping results. Whole genome sequencing was conducted on ten 33F-1 pneumococcal isolates. Initially, sequence reads were used for molecular serotyping by PneumoCaT. Phenotypic typing of 33F-1 isolates was then performed using the Quellung reaction and latex agglutination. Genome assemblies were used in phylogenetic analyses of each gene in the capsular locus to investigate genetic divergence. All ten pneumococcal isolates with the 33F-1 cps locus typed as 33F by Quellung and latex agglutination. Unlike the reference 33F capsule locus sequence, DNA microarray and PneumoCaT analyses found that 33F-1 pneumococci lack the wcjE gene, and instead contain wcyO with a frameshift mutation. Phylogenetic analyses found the wzg, wzh, wzd, wze, wchA, wciG and glf genes in the 33F-1 cps locus had higher DNA sequence similarity to homologues from other serotypes than to the 33F reference sequence. We have discovered a novel genetic variant of serotype 33F, which lacks wcjE and contains a wcyO pseudogene. This finding adds to the understanding of molecular epidemiology of pneumococcal serotype diversity, which is poorly understood in low and middle-income countries.

Effect of ten-valent pneumococcal conjugate vaccine introduction on pneumococcal carriage in Fiji: results from four annual cross-sectional carriage surveys.

BACKGROUND: The indirect effects of pneumococcal conjugate vaccines (PCVs) are mediated through reductions in carriage of vaccine serotypes. Data on PCVs in Asia and the Pacific are scarce. Fiji introduced the ten-valent PCV (PCV10) in 2012, with a schedule consisting of three priming doses at 6, 10, and 14 weeks of age and no booster dose (3 + 0 schedule) without catch-up. We investigated the effects of PCV10 introduction using cross-sectional nasopharyngeal carriage surveys. METHODS: We did four annual carriage surveys (one pre-PCV10 and three post-PCV10) in the greater Suva area in Fiji, during 2012-15, of 5-8-week-old infants, 12-23-month-old children, 2-6-year-old children, and their caregivers (total of 8109 participants). Eligible participants were of appropriate age, had axillary temperature lower than 37°C, and had lived in the community for at least 3 consecutive months. We used purposive quota sampling to ensure a proper representation of the Fiji population. Pneumococci were detected by real-time quantitative PCR, and molecular serotyping was done with microarray. FINDINGS: 3 years after PCV10 introduction, vaccine-serotype carriage prevalence declined, with adjusted prevalences (2015 vs 2012) of 0·56 (95% CI 0·34-0·93) in 5-8-week-old infants, 0·34 (0·23-0·49) in 12-23-month-olds, 0·47 (0·34-0·66) in 2-6-year-olds, and 0·43 (0·13-1·42) in caregivers. Reductions in PCV10 serotype carriage were evident in both main ethnic groups in Fiji; however, carriage of non-PCV10 serotypes increased in Indigenous Fijian infants and children. Density of PCV10 serotypes and non-PCV10 serotypes was lower in PCV10-vaccinated children aged 12-23 months than in PCV10-unvaccinated children of the same age group (PCV10 serotypes -0·56 [95% CI -0·98 to -0·15], p=0·0077; non-PCV10 serotypes -0·29 [-0·57 to -0·02], p=0·0334). INTERPRETATION: Direct and indirect effects on pneumococcal carriage post-PCV10 are likely to result in reductions in pneumococcal disease, including in infants too young to be vaccinated. Serotype replacement in carriage in Fijian children, particularly Indigenous children, warrants further monitoring. Observed changes in pneumococcal density might be temporal rather than vaccine related. FUNDING: Department of Foreign Affairs and Trade of the Australian Government through the Fiji Health Sector Support Program; Victorian Government's Operational Infrastructure Support Program; Bill & Melinda Gates Foundation.

Capsular Serotyping of Streptococcus pneumoniae by latex agglutination.

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the 'gold standard' Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. This method of production and quality control may also be suitable for other testing purposes.

Production of latex agglutination reagents for pneumococcal serotyping.

BACKGROUND: The current 'gold standard' for serotyping pneumococci is the Quellung test. This technique is laborious and requires a certain level of training to correctly perform. Commercial pneumococcal latex agglutination serotyping reagents are available, but these are expensive. In-house production of latex agglutination reagents can be a cost-effective alternative to using commercially available reagents. This paper describes a method for the production and quality control (QC) of latex reagents, including problem solving recommendations, for pneumococcal serotyping. RESULTS: Here we describe a method for the production of latex agglutination reagents based on the passive adsorption of antibodies to latex particles. Sixty-five latex agglutination reagents were made using the PneuCarriage Project (PCP) method, of which 35 passed QC. The other 30 reagents failed QC due to auto-agglutination (n=2), no reactivity with target serotypes (n=8) or cross-reactivity with non-target serotypes (n=20). Dilution of antisera resulted in a further 27 reagents passing QC. The remaining three reagents passed QC when prepared without centrifugation and wash steps. Protein estimates indicated that latex reagents that failed QC when prepared using the PCP method passed when made with antiserum containing ≤ 500 µg/ml of protein. Sixty-one nasopharyngeal isolates were serotyped with our in-house latex agglutination reagents, with the results showing complete concordance with the Quellung reaction. CONCLUSIONS: The method described here to produce latex agglutination reagents allows simple and efficient serotyping of pneumococci and may be applicable to latex agglutination reagents for typing or identification of other microorganisms. We recommend diluting antisera or removing centrifugation and wash steps for any latex reagents that fail QC. Our latex reagents are cost-effective, technically undemanding to prepare and remain stable for long periods of time, making them ideal for use in low-income countries.