Pneumococcal Carriage In Children Research Articles

A low-cost culture- and DNA extraction-free method for the molecular detection of pneumococcal carriage in saliva.

Molecular methods have improved the sensitivity of the detection of pneumococcal carriage in saliva. However, they typically require sample culture enrichment and nucleic acid extraction prior to performing the detection assay and may limit scalability for extensive surveillance of pneumococcus, particularly in low-resource settings. We evaluated the performance of a DNA-extraction-free method for the detection of pneumococcus in saliva. We developed a streamlined qPCR-based protocol for the detection of pneumococcus, omitting culture enrichment and DNA extraction. Using saliva samples collected from children attending childcare centers (New Haven, CT, USA), we evaluated the detection of pneumococcus using saliva lysates as compared to purified DNA extracted from culture-enriched aliquots of the paired samples using qPCR targeting the pneumococcal piaB gene. Of the 759 saliva samples tested from 92 children [median age 3.65 years; IQR (2.46-4.78)], pneumococcus was detected in 358 (47.2%) saliva lysates prepared using the extraction-free protocol and in 369 (48.6%) DNA extracted from culture-enriched samples. We observed near-perfect agreement between the two protocols (Cohen's kappa: 0.92; 95% CI: 0.90-0.95). Despite a high correlation between CT values generated by the two methods (r = 0.93, P < 0.0001), the CT values generated from saliva lysates were higher (lower concentration) than those from culture-enriched samples (ΔCT = 6.69, P < 0.00001). The cost of detecting pneumococcus using saliva lysates was at least fivefold lower (US$2.53) compared to the cost of the culture-enriched method (range: US$13.60-US$19.46). For pneumococcal carriage surveillance in children, our findings suggest that a DNA extraction-free approach may offer a cost-effective alternative to the resource-intensive culture-enrichment method.IMPORTANCESurveillance for carriage of pneumococcus is a key component of evaluating the performance of pneumococcal vaccines and informing new vaccination strategies. To improve the scalability of pneumococcal carriage surveillance, we show that molecular detection of pneumococcus in saliva from children can be performed without culture enrichment and DNA extraction. Our findings show that using the extraction-free method can improve surveillance efforts for pneumococcal carriage in children, overcoming the resource-intensive hurdle that comes with the use of molecular methods, particularly in low-resource settings.

Pneumococcal carriage and disease in adults in England 2011-2019: the importance of adults as a reservoir for pneumococcus in communities.

Pneumococcal carriage in children has been extensively studied, but carriage in healthy adults and its relationship to invasive pneumococcal disease (IPD) is less understood. Nasal wash samples from adults without close contact with young children (Liverpool, UK), 2011-2019, were cultured, and culture-negative samples tested by PCR. Pneumococcal carriage in adults 18-44 years was compared with carriage among PCV-vaccinated children 13-48 months (nasopharyngeal swabs, Thames Valley, UK) and IPD data for England for the same ages for 2014-2019. Age-group specific serotype invasiveness was calculated and used with national IPD data to estimate carriage serotype distributions for adults aged 65+ years. In total 98 isolates (97 carriers) were identified from 1,631 adults aged 18+ years (age and sex standardized carriage prevalence 6.4%), with only three identified solely by PCR. Despite different carriage and IPD serotype distributions between adults and children, serotype invasiveness was highly correlated (R=0.9). Serotypes 3, 37 and 8 represented a higher proportion of adult carriage than expected from direct low-level transmission from children to adults. The predicted carriage serotype distributions for 65+ years aligned more closely with the carriage serotype distribution for young adults than young children. The nasal wash technique is highly sensitive; additional benefit of PCR is limited. Comparison of carriage serotype distributions suggests some serotypes may be circulating preferentially within these specific young adults. Our data suggest that for some serotypes carried by adults 65+ years, other adults may be an important reservoir for transmission. Age groups such as older children should also be considered.

Molecular epidemiology of pneumococcal carriage in children from Seville, following implementation of the PCV13 immunization program in Andalusia, Spain

IntroductionThe 13-valent pneumococcal conjugate vaccine (PCV13) universal vaccination programme was introduced in December 2016 in Andalusia. MethodsA cross-sectional study was conducted on the molecular epidemiology of pneumococcal nasopharyngeal colonization. A total of 397 healthy children were recruited from primary healthcare centres in Seville for the periods 1/4/2018 to 28/2/2020 and 1/11/2021 to 28/2/2022 (PCV13 period). Data from a previous carriage study conducted among healthy and sick children from 1/01/2006 to 30/06/2008 (PCV7 period), were used for comparison of serotype/genotype distributions and antibiotic resistance rates. ResultsOverall, 76 (19%) children were colonized with S. pneumoniae during the PCV13 period and there were information available from 154 isolates collected during the PCV7 period. Colonization with PCV13 serotypes declined significantly in the PCV13 period compared with historical controls (11% vs 38%, p = 0.0001), being serotypes 19F (8%), 3 (1%) and 6B (1%) the only circulating vaccine types. Serotypes 15B/C and 11A were the most frequently identified non-PCV13 serotypes during the PCV13 period (14% and 11%, respectively); the later one increased significantly between time periods (p = 0.04). Serotype 11A was exclusively associated in the PCV13 period with ampicillin-resistant variants of the Spain9V-ST156 clone (ST6521 and genetically related ST14698), not detected in the preceding period. ConclusionsThere was a residual circulation of vaccine types following PCV13 introduction, apart from serotype 19F. Serotype 11A increased between PCV13 and PCV7 periods due to emergence and clonal expansion of ampicillin-resistant genotype ST6521.

A spitting image: molecular diagnostics applied to saliva enhance detection of Streptococcus pneumoniae and pneumococcal serotype carriage.

Despite strong historical records on the accuracy of saliva testing, oral fluids are considered poorly suited for pneumococcal carriage detection. We evaluated an approach for carriage surveillance and vaccine studies that increases the sensitivity and specificity of pneumococcus and pneumococcal serotype detection in saliva samples. Quantitative PCR (qPCR)-based methods were applied to detect pneumococcus and pneumococcal serotypes in 971 saliva samples collected from 653 toddlers and 318 adults. Results were compared with culture-based and qPCR-based detection in nasopharyngeal samples collected from children and in nasopharyngeal and oropharyngeal samples collected from adults. Optimal C q cut-offs for positivity in qPCRs were determined via receiver operating characteristic curve analysis and accuracy of different approaches was assessed using a composite reference for pneumococcal and for serotype carriage based on isolation of live pneumococcus from the person or positivity of saliva samples determined with qPCR. To evaluate the inter-laboratory reproducibility of the method, 229 culture-enriched samples were tested independently in the second center. In total, 51.5% of saliva samples from children and 31.8% of saliva samples from adults were positive for pneumococcus. Detection of pneumococcus by qPCR in culture-enriched saliva exhibited enhanced sensitivity and higher agreement with a composite reference compared to diagnostic culture of nasopharyngeal samples in children (Cohen's κ: 0.69-0.79 vs. 0.61-0.73) and in adults (κ: 0.84-0.95 vs. 0.04-0.33) and culture of oropharyngeal samples in adults (κ: 0.84-0.95 vs. -0.12-0.19). Similarly, detection of serotypes with qPCR in culture-enriched saliva exhibited enhanced sensitivity and higher agreement with a composite reference compared to nasopharyngeal culture in children (κ: 0.73-0.82 vs. 0.61-0.73) and adults (κ: 0.90-0.96 vs. 0.00-0.30) and oropharyngeal culture in adults (κ: 0.90-0.96 vs. -0.13 to 0.30). However, results of qPCRs targeting serotype 4, 5, and 17F and serogroups 9, 12, and 35 were excluded due to assays' lack of specificity. We observed excellent quantitative agreement for qPCR-based detection of pneumococcus between laboratories. After exclusion of serotype/serogroup-specific assays with insufficient specificity, moderate agreement (κ 0.68, 95% CI 0.58-0.77) was observed. Molecular testing of culture-enriched saliva samples improves the sensitivity of overall surveillance of pneumococcal carriage in children and adults, but limitations of qPCR-based approaches for pneumococcal serotypes carriage detection should be considered.

90Factors associated with pneumococcal carriage in children and adults in Fiji, using four cross-sectional surveys

Abstract Background Pneumococcal disease is preceded by carriage of pneumococci. We describe factors associated with pneumococcal nasopharyngeal carriage in Fiji, using data from annual (2012-2015) cross-sectional surveys, pre- and post-introduction of ten-valent pneumococcal conjugate vaccine (PCV10). Methods Infants (5-8 weeks), toddlers (12-23 months), children (2-6 years), and their caregivers participated. Pneumococci were detected using lytA qPCR, with molecular serotyping by microarray. We used logistic regression to determine predictors of carriage and density. Results There were 8,109 participants. Pneumococcal carriage was associated with: years post-PCV10 introduction (global P&amp;lt;0.001), iTaukei ethnicity (aOR 2.74 [95% CI 2.17-3.45] P&amp;lt;0.001); young age (global P&amp;lt;0.001); urban residence (aOR 1.45 [95% CI 1.30-2.57] P&amp;lt;0.001); living with &amp;gt;2 children &amp;lt;5 years (aOR 1.42 [95% CI 1.27-1.59] P&amp;lt;0.001); poverty (aOR 1.44 [95% CI 1.28-1.62] P&amp;lt;0.001); and upper respiratory tract infection (URTI) symptoms (aOR 1.77 [95% CI 1.57-2.01] P&amp;lt;0.001). Factors associated with PCV10 and non-PCV10 carriage were similar to those associated with overall carriage. Additionally, PCV10 carriage was associated with PCV10 vaccination (0.58 [95% CI 0.41-0.82] P=0.002) and cigarette smoke exposure (aOR 1.21 [95% CI 1.02-1.43] P=0.031. Non-PCV10 carriage was not associated with years post-PCV10 introduction. Conclusions Introduction of PCV10 reduced the odds of overall and PCV10 pneumococcal carriage in Fiji. ITaukei ethnicity was positively associated with carriage after adjustment for PCV10. Key messages PCV10 introduction was associated with reduced odds of overall and PCV10 pneumococcal carriage in Fiji. Despite high and similar PCV10 coverage rates, iTaukei ethnicity is positively associated with pneumococcal carriage, compared with Fijian of Indian Descent populations.

Direct and indirect effects of 13-valent pneumococcal conjugate vaccine on pneumococcal carriage in children hospitalised with pneumonia from formal and informal settlements in Mongolia: an observational study.

BackgroundWithin Ulaanbaatar, Mongolia, risk factors for pneumonia are concentrated among children living in informal settlements comprised of temporary shelters (gers). We used pneumococcal carriage surveillance among children from formal and informal settlements hospitalised with pneumonia to evaluate the direct and indirect effects of 13-valent pneumococcal conjugate vaccine (PCV13) against vaccine-type (VT) pneumococcal carriage following a phased introduction of PCV13.MethodsWe enrolled and collected nasopharyngeal swabs from children 2-59 months of age presenting to hospital. Pneumococci were detected using lytA qPCR and serotyped using microarray on a random monthly selection of swabs between November 2015 and March 2019 from two districts in Ulaanbaatar. PCV13 status was determined using written records. We quantified the associations between individual PCV13 status (direct effects) and district-level PCV13 coverage (indirect effects) and VT carriage using generalised estimating equations and explored interactions by settlement type.FindingsA total of 1 292 swabs from 6 046 participants were tested for pneumococci. Receipt of PCV13 and increasing PCV13 coverage independently reduced the risk of VT carriage. For each percent increase in PCV13 coverage, the adjusted odds of VT carriage decreased by 1•0% (OR 95% CI 0•983-0•996; p=0•001), with a predicted decrease in VT carriage rate from 29•1% to 13•1% as coverage reached 100%. There was a trend towards a slower decline within informal settlements (p=0•100). Adjusted PCV13 vaccine effectiveness against VT carriage was 39•1% (95% CI 11•4-58•1%, p=0•009).InterpretationSubstantial indirect effects were observed following PCV13 introduction, including among children living within informal settlements.FundingBill & Melinda Gates Foundation; Gavi, the Vaccine Alliance.

Indirect effects of 13-valent pneumococcal conjugate vaccine on pneumococcal carriage in children hospitalised with acute respiratory infection despite heterogeneous vaccine coverage: an observational study in Lao People’s Democratic Republic

IntroductionEmpiric data on indirect (herd) effects of pneumococcal conjugate vaccines (PCVs) in settings with low or heterogeneous PCV coverage are limited. The indirect effects of PCV, which benefits both vaccinated...

A Narrative Review of Pneumococcal Disease in Children in the Philippines.

This narrative review describes the epidemiology of invasive pneumococcal diseases, nasopharyngeal carriage, and antibiotic resistance of Streptococcus pneumoniae serotypes, and vaccination coverage in children in the Philippines. Epidemiological data show that, despite the availability of the free-of-cost 13-valent pneumococcal conjugate vaccine for infants as part of the National Immunization Program, the burden of pneumococcal disease in young children remains high in the Philippines. The significant variability in data reported between studies highlights an urgent need for active and comprehensive disease surveillance for more accurate estimates of pneumococcal disease in the country. Although data from 2001 to 2013 show high rates of pneumococcal carriage in children in the Philippines aged < 5 years, contemporary data are lacking, again emphasizing the need for active surveillance programs. The introduction of pneumococcal conjugate vaccines has resulted in substantial declines in disease caused by pneumococcal serotypes included in the vaccines, but the emergence of pneumococcal disease due to nonvaccine serotypes is an ongoing concern. Surveillance of actively circulating serotypes is critical to better understand vaccine coverage. Antimicrobial resistance of S. pneumoniae remains a significant threat to public health worldwide; data regarding antibiotic resistance in young children in the Philippines are limited, but reports generally show low rates of antibiotic resistance in this group. National immunization rates have increased in recent years, yet many individuals are still unprotected from pneumococcal disease. Overall, there is a critical need for contemporary and accurate disease surveillance in the Philippines. Such data would provide better estimates of pneumococcal disease incidence, serotype distribution, and antibiotic resistance to better inform vaccination strategies and to ensure that children in the Philippines are best protected against pneumococcal disease.

Pneumococcal carriage in children with COVID-19

ABSTRACT Background: SARS-CoV-2 is the new virus, and Streptococcus pneumoniae is one of the most important pathogens affecting humans. However, we do not yet know whether these microorganisms interact. Thus, we aimed to evaluate the relationship between Streptococcus pneumoniae and SARS-CoV-2 in pediatric patients. Methods: This study was conducted retrospectively by means of medical records of pediatric patients who were tested for SARS-CoV-2 between March 11 and June 04, 2020, in the University of Health Sciences, Ankara Educating and Training Hospital and Hacettepe University Faculty of Medicine. Results: We evaluated 829 pediatric patients for S. pneumoniae and SARS-CoV-2 from their nasopharyngeal specimen. Of 115 children positive for SARS-CoV-2, 32.2% had a positive S. pneumoniae test, whereas of 714 children negative for SARS-CoV-2, 14.1% had a positive S. pneumoniae test (p < .01). We compared patients with positive vs. negative SARS-CoV-2 tests according to S. pneumoniae positivity There were no statistically significant differences in terms of gender, underlying disease, fever, cough, leukocytosis, lymphopenia, increased CRP, increased procalcitonin, findings of chest x-ray, severity of disease, and treatment. Conclusion: The nasopharyngeal S. pneumoniae carriage rate in patients with COVID-19 was higher than in non-infected children, while S. pneumoniae carriage did not affect the course of COVID-19 disease. Pneumococcal vaccination is significant, such that we do not know the outcomes of increased pneumococcal carriage for the upcoming months of pandemic.

Multicenter study of pneumococcal carriage in children 2 to 4 years of age in the winter seasons of 2017-2019 in Irbid and Madaba governorates of Jordan.

Streptococcus pneumoniae is one of the leading causes of death worldwide. It disseminates through colonizers and causes serious infections. Aims of this study are to determine pneumococcal carriage rate, resistance, serotype distribution, and coverage of pneumococcal conjugate vaccines from children attending day care centers from Irbid and Madaba in Jordan. Nasopharyngeal swabs were collected from day care centers (DCCs) of healthy Jordanian children 2-4 years of age from four regions of Madaba (n = 596), and from eastern Irbid (n = 423). Swabs were cultivated on Columbia blood agar base supplemented with 5% sheep blood and incubated for 18-24 hours at 37°C with 5% CO2. Alpha-hemolytic isolates were tested for optochin sensitivity and bile solubility for identification. Isolates were analyzed for antimicrobial susceptibility by the Vitek2 system and E-test (BioMérieux). Serotyping was performed using the Neufeld Quellung method. A total of 341 pneumococcal strains were isolated from 1019 nasopharyngeal (NP) samples of healthy children attending DCCs for two winter seasons from 2017-2019. Carriage rate in eastern Irbid for both seasons was 29.6% and for Madaba 37.9%. Resistance rates for Irbid and Madaba, respectively, were as follows: Penicillin (86.3%; 94.4%), erythromycin (57.0%; 78.2%), clindamycin (30.8%; 47.2%), trimethoprim-sulfamethoxazole (68.6%; 86.6%), and tetracycline (45.7%; 51.9%). Predominant serotypes for Irbid were 19F (20.8%), 23F (12.0%), 6A (10.4%), and 6B (9.6%); whereas for Madaba were 19F (24.5%), 14 (7.4%), 6A (6.9%) and 23F (6.5%). Serotype coverage of the thirteen valent pneumococcal conjugate vaccine (PCV13) was about 65% for both regions. Over 96% of isolates with PCV13 serotypes in this study were resistant to penicillin with the exception of serotypes 3 and 5. As a conclusion resistance and carriage rates among the age group 2 to 4 years reached an alarming rate especially among vaccine types, which can be controlled by pneumococcal conjugate vaccination strategies.

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

BackgroundNasopharyngeal 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. MethodsWe 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. FindingsOne 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. ConclusionThis 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 children and their household contacts six years after introduction of the 13-valent pneumococcal conjugate vaccine in England.

BackgroundIn April 2010, 13-valent pneumococcal conjugate vaccine (PCV13) replaced PCV7 in the infant immunisation schedule in England and Wales. Despite limited serotype replacement in invasive pneumococcal disease (IPD) during the first four post-PCV13 years, non-vaccine type (NVT) IPD increased substantially in 2014/15. We undertook a carriage study in 2015/16 to help understand the reasons for this increase.Methods and findingsFamilies with a child aged <5 years attending a participating general practice in Gloucestershire or Hertfordshire were invited to provide nasopharyngeal swabs from all consenting members. Swabs from 650 individuals (293 under five, 73 five to twenty and 284 >twenty years) were cultured and serotyped for Streptococcus pneumoniae. Results were compared with those from three previous household studies conducted in the same populations between 2001 to 2013, and with the serotypes causing IPD to estimate case-carrier ratios (CCRs). Overall carriage prevalence did not differ between the four carriage studies with reductions in vaccine-type carriage offset by increases in NVT carriage. While no individual NVT serotype showed an increase in CCR from 2012/13, the composition of the serotypes comprising the NVT group differed such that the overall CCR of the NVT group had significantly increased since 2012/13. Carriage of two PCV13 serotypes, 3 and 19A, was found in 2015/16 (3/650 = 0.5% and 2/650 = 0.3% respectively) with no overall reduction in carriage prevalence of PCV13-7 serotypes since 2012/13, though 6C prevalence, a vaccine-related serotype, had reduced from 1.8% in 2012/13 to 2/648 (0.3%) in 2015/16, p = 0.013.ConclusionsThere was continuing evolution in carried NVTs six years after PCV13 introduction which, in addition to being vaccine-driven, could also reflect natural secular changes in certain NVTs. This poses challenges in predicting future trends in IPD. Elimination of carriage and disease due to serotypes 3 and 19A may not be achieved by PCV13.

Pneumococcal Nasopharyngeal Carriage in Young Healthy Children After Pneumococcal Conjugate Vaccine in Turkey.

Aims: To determine the prevalence of nasopharyngeal carriage of Streptococcus pneumoniae in healthy children aged 0-6 years who were vaccinated with pneumococcal conjugate vaccine.Methods: This cross-sectional study was conducted on 150 healthy Turkish children between 1 month and 6 years of age. Serotyping was performed and risk factors of carriage were evaluated.Results: The overall carriage rate was 14%. Vaccine type serotypes were determined in 17 (12.6%) children who received full-dose PCV13 vaccine. The highest carriage rate was observed among children younger than 24 months (76.2%). In multivariate analysis, respiratory infection in recent months, age, attendance at a day-care center and antibiotic usage were not statistically significant risk factors for carriage. Overall, S. pneumoniae strains were considered as penicillin susceptible and antimicrobial resistance was limited.Conclusion: We observed a low rate of pneumococcal carriage in children after PCV13 implementation compared with that of children receiving PCV7. Although it was reduced, vaccine serotype colonization in PCV13-vaccinated children remains persistent.

Pneumococcal Carriage in Children under Five Years in Uganda-Will Present Pneumococcal Conjugate Vaccines Be Appropriate?

BackgroundPneumonia is the major cause of death in children globally, with more than 900,000 deaths annually in children under five years of age. Streptococcus pneumoniae causes most deaths, most often in the form of community acquired pneumonia. Pneumococcal conjugate vaccines (PCVs) are currently being implemented in many low-income countries. PCVs decrease vaccine-type pneumococcal carriage, a prerequisite for invasive pneumococcal disease, and thereby affects pneumococcal disease and transmission. In Uganda, PCV was launched in 2014, but baseline data is lacking for pneumococcal serotypes in carriage.ObjectivesTo study pneumococcal nasopharyngeal carriage and serotype distribution in children under 5 years of age prior to PCV introduction in UgandaMethodsThree cross-sectional pneumococcal carriage surveys were conducted in 2008, 2009 and 2011, comprising respectively 150, 587 and 1024 randomly selected children aged less than five years from the Iganga/Mayuge Health and Demographic Surveillance Site. The caretakers were interviewed about illness history of the child and 1723 nasopharyngeal specimens were collected. From these, 927 isolates of S. pneumoniae were serotyped.ResultsOverall, the carriage rate of S. pneumoniae was 56% (957/1723). Pneumococcal carriage was associated with illness on the day of the interview (OR = 1.50, p = 0.04). The most common pneumococcal serotypes were in descending order 19F (16%), 23F (9%), 6A (8%), 29 (7%) and 6B (7%). One percent of the strains were non-typeable. The potential serotype coverage rate for PCV10 was 42% and 54% for PCV13.ConclusionAbout half of circulating pneumococcal serotypes in carriage in the Ugandan under-five population studied was covered by available PCVs.

Real-time Reverse Transcription Polymerase Chain Reaction Using Total RNA Extracted from Nasopharyngeal Aspirates for Detection of Pneumococcal Carriage in Children

목적: 폐렴구균은 주요 비인두 상재균으로, 주위 조직을 침범하여 침습성 감염을 일으킬 수 있어 보균 율에 대한 감시가 중요하다. 본 연구에서는 임상에서 비인두 흡인물로부터 추출하고 남은 RNA를 이용 하여 폐렴구균을 확인할 수 있는 실시간 중합효소 연쇄반응(real-time reverse transcription poly-merase chain reaction [RT-PCR])법을 구축하고, 보균율 측정에 있어서의 정확성과 이점을 확인하고자 하였다. 방법: 2014년 9월부터 10월까지 중앙대학교병원에 입원하여 호흡기 바이러스 RT-PCR 검사를 시행 받은 18세 이하의 소아들로부터 비인두 흡인물을 채취하였다. 먼저 배양법과 genomic DNA (gDNA) 를 이용한 real-time PCR을 시행하여 폐렴구균 검출률의 정확성을 확인하였다. 이 중 처음 20개의 검체 를 이용하여, 고전적인 배양법과 gDNA를 이용한 real-time PCR, 그리고 RNA를 이용한 real-time RT-PCR법을 시행하고 이를 비교 분석하였다. 결과: 총 157개의 검체에서 시행한 real-time PCR 검사는 기존의 배양검사와 일치율이 0.922 (P<0.01, Fisher exact test)로 매우 높았다. 배양검사에서 음성인 133개의 검체는 real-time PCR에서도 모두 음성을 보였다. 24개의 배양 양성 검체 중 21개의 검체는 real-time PCR에서도 양성이었지만, 나머지 검체는 음성 결과를 보였다. 20개의 검체에서 시행한 real-time RT-PCR 검사는 1개 검체를 제외 하고 배양법 및 real-time PCR과 결과가 일치하였다. 한편, 배양법을 시행하고 결과를 확인하기까지 는 총 26.5시간, real-time RT-PCR 검사에는 총 4.5시간이 소요되었다. 결론: 본 연구는 비인두 집락균 확인을 위한 real-time RT-PCR법의 확립과, 폐렴구균 보균율 측정에 있어서의 real-time RT-PCR 검사의 정확성 및 편의성을 보여주었다. Real-time RT-PCR 검사법 은 주요 세균들의 보균율 연구에 있어서 시간과 노력을 줄일 수 있는 좋은 방법이며, 폐렴구균의 역학 자료 수집에 큰 도움이 될 것으로 기대한다.

Association of Pneumococcal Carriage and Expression of Foxp3+ Regulatory T Cells and Th17 Cells in the Adenoids of Children

Background: Pneumococcal carriage in the nasopharynx is a primary means of transmission and a necessary prerequisite for pneumococcal disease. Objectives: We analyzed the relationship between expressions of Foxp3+ regulatory T (T_reg) cells and Th17 cells, and pneumococcal carriage in the adenoids of children who were either positive or negative for pneumococci. Methods: We collected adenoidal tissue and nasopharyngeal swab samples from children undergoing an adenoidectomy. Adenoidal mononuclear cells were isolated, cultured and then stimulated with culture concentrated supernatant (CCS) obtained from a D39 bacterial strain. Results: Foxp3+ T_reg cells were upregulated and Th17 cells were downregulated in populations of adenoidal mononuclear cells obtained from the pneumococcus-positive group. Following CCS stimulation, the increment in Foxp3+ T_reg cells in the pneumococcus-positive group was significantly greater than that in the pneumococcus-negative group, while the increment in Th17 cells was less as compared to that in the pneumococcus-negative group. These results were consistent with variations in levels of Foxp3 mRNA and retinoic acid receptor-related orphan receptor-γt mRNA in adenoidal mononuclear cells. Levels of IL-17A and IL-6 in adenoid tissue were higher in the pneumococcus-negative group, and the levels of TGF-β in adenoid tissue were lower in the pneumococcus-negative group compared to the pneumococcus-positive group. Pneumococcal carriage in children was closely associated with the expressions of Foxp3+ T_reg and Th17 cells in the adenoid. Conclusion: Upregulation of Foxp3+ T_reg cells might downregulate the production of Th17 cells in the adenoid, resulting in decreased scavenging of Streptococcus pneumoniae and chronic pneumococcal carriage.

Epidemiology of pneumococcal carriage in children under five years of age in Accra, Ghana

Background: Although the majority of pneumococcal infections occur in the developing world, pneumococcal epidemiology is poorly understood in these settings. The aim of the study was to investigate the epidemiology of pneumococcal carriage among children younger than 5 years at a paediatric healthcare centre in Ghana. Method: Four-hundred and twenty-three children were randomly sampled and nasopharyngeal specimens were collected from them. The specimens were cultured for Streptococcus pneumoniae, and the isolates were subjected to antibiotic susceptibility testing and serotyping by latex agglutination. Epidemiological data on demographic and clinical features of the study subjects were collected. Results: The prevalence of pneumococcal carriage was 48.9% (207/422), with age groups 43–48 months having the highest carriage prevalence. In the multivariate analysis, pneumococcal carriage was significantly associated with runny nose (odds ratio = 1.9, p = 0.003) and day-care attendance (odds ratio = 1.5, p = 0.04). No pneumococcal resistance was observed for ceftriaxone, while the prevalence of resistance to the other antibiotics tested was: cotrimoxazole 100%, ampicillin 88%, tetracycline 78%, penicillin 63% and erythromycin 24%. Fourteen different pneumococcal serogroups/serotypes were identified and serogroup 6 was the most prevalent (30%), followed by serotype 19 (20%). Conclusions: We conclude that pneumococcal carriage among the study children is high and the carried strains have a high level of resistance (> 50%) to several antibiotics. Ceftriaxone is a suitable antibiotic for treating pneumococcal infections in Ghana, and the use of this antibiotic coupled with the pneumococcal vaccination is expected to significantly reduce the burden of pneumococcal disease in the country.

A cross-sectional observational study of pneumococcal carriage in children, their parents, and older adults following the introduction of the 7-valent pneumococcal conjugate vaccine.

Using nasopharyngeal carriage as a marker of vaccine impact, pneumococcal colonization and its relation to invasive disease were examined in children, their parents, and older adults in the United Kingdom following introduction of 7-valent pneumococcal conjugate vaccine (PCV7) and prior to 13-valent pneumococcal conjugate vaccine (PCV13).A cross-sectional observational study was conducted, collecting nasopharyngeal swabs from children aged 25 to 55 months who had previously received 3 doses of PCV7, their parents, and adults aged ≥65 years. Pneumococcal serotyping was conducted according to World Health Organization guidelines with nontypeable isolates further analyzed by molecular serotyping. A national invasive disease surveillance program was conducted throughout the corresponding period.Pneumococcus was isolated from 47% of children, 9% of parents, and 2.2% of older adults. For these groups, the percentage of serotypes covered by PCV7 were 1.5%, 0.0%, and 15.4%, with a further 20.1%, 44.4%, and 7.7% coverage added by those in PCV13. In each group, the percentage of disease due to serotypes covered by PCV7 were 1.0%, 7.4% and 5.1% with a further 65.3%, 42.1%, and 61.4% attributed to those in PCV13.The prevalence of carriage is the highest in children, with direct vaccine impact exemplified by low carriage and disease prevalence of PCV7 serotypes in vaccinated children, whereas the indirect effects of herd protection are implied by similar observations in unvaccinated parents and older adults.

Pneumococcal carriage in children and adults two years after introduction of the thirteen valent pneumococcal conjugate vaccine in England

Background/AimsIn April 2010 the 7-valent pneumococcal conjugate vaccine (PCV7) was replaced by the 13-valent PCV. We investigated pneumococcal carriage in children eligible for PCV7 or PCV13 and their household contacts. MethodsEligible families in Hertfordshire and Gloucester were identified and a nasopharyngeal swab obtained from consenting household members between July 2012 and March 2013. Samples were cultured for Streptococcus pneumoniae and serotyped by standard methods. For each serotype the ratio of its prevalence in invasive pneumococcal disease (IPD) to its carriage prevalence (case:carrier ratio, CCR) was calculated. Results were compared with previous carriage studies in 2001/2002 and 2008/2009, before and after PCV7 introduction. Results217 households were included. Among <5-year olds 47.7% (95% confidence interval 41.8–53.5) were carrying a pneumococcus compared with 51.0% (95% CI: 44.0–58.0) in 2008/2009 and 48.4% (95% CI: 44.1–52.7) in 2001/2002. The odds of carrying a PCV7 serotype was significantly reduced in 2008/2009 (0.07, 95% CI: 0.03–0.16) and 2012/2013 (0.01 95% CI: 0.00–0.07) relative to 2001/2002, while the odds of carrying any of the extra six PCV13 serotypes increased after PCV7 introduction (1.38, 95%CI: 0.73–2.59) but declined significantly after PCV13 introduction (0.05, 95%CI: 0.01–0.37). The CCRs for the frequently carried serotypes were relatively low, with the highest CCR observed for serotypes 7F, 19A, 3, 8, and 33F. Across the three carriage studies, CCR estimates were stable for nearly all serotypes. ConclusionCarriage of additional PCV13 serotypes has rapidly reduced post-PCV13 introduction in both vaccinated and unvaccinated individuals with a continued decline in transmission of PCV7 serotypes. Carriage rates in children remain unchanged, but the low CCRs of replacing serotypes would be expected to further reduce overall IPD across all age groups.

Immune responses to pneumococcal pilus RrgA and RrgB antigens and their relationship with pneumococcal carriage in humans

Pneumococcal pilus antigens are shown to be important in pneumococcal pathogenesis and induce protective immunity in animal studies, but data in humans are limited. We aimed to investigate serum and mucosal immune responses to pilus-1 proteins (RrgA and RrgB) and their relationship with pneumococcal carriage in humans. Serum and salivary antibodies to RrgA and RrgB in children and adults were analysed by ELISA and immunoblotting. Induction of B cell antibody responses to RrgA and RrgB in nasopharynx-associated lymphoid tissue was studied by ELISpot assay following stimulation with pneumococcal culture supernatants containing pilus proteins. Significant levels of serum anti-RrgA and -RrgB antibodies were observed, and anti-RrgA antibody appeared to develop earlier in childhood. Importantly, anti-RrgA IgG titres in both serum and saliva were shown to be higher in culture-negative children than in those who were culture-positive for Streptococcus pneumoniae. Stimulation of adenotonsillar cells with pneumococcal culture supernatant induced significant RrgA- and RrgB-specific antibody secreting cells and antibody production. Pneumococcal pilus antigens, particularly RrgA, seem to induce significant serum and mucosal antibody responses that may contribute to natural immunity against pneumococcal carriage in children.