Nasopharyngeal carriage of Streptococcus pneumoniae among children <5 years of age in Indonesia prior to pneumococcal conjugate vaccine introduction.

Pneumococcal conjugate vaccines (PCVs) prevent nasopharyngeal colonization with vaccine serotypes of Streptococcus pneumoniae, leading to reduced transmission of pneumococci and stronger population-level impact of PCVs. In 2017 we conducted a cross-sectional pneumococcal carriage study in Indonesia among children aged <5 years before 13-valent PCV (PCV13) introduction. Nasopharyngeal swabs were collected during visits to community integrated health service posts at one peri-urban and one rural study site. Specimens were analyzed by culture, and isolates were serotyped using sequential multiplex polymerase chain and Quellung reaction. Antibiotic susceptibility was performed by broth microdilution method. We enrolled 1,007 children in Gunungkidul District, Yogyakarta (peri-urban) and 815 in Southwest Sumba, East Nusa Tenggara (rural). Pneumococcal carriage prevalence was 30.9% in Gunungkidul and 87.6% in Southwest Sumba (combined: 56.3%). PCV13 serotypes (VT) carriage was 15.0% in Gunungkidul and 52.6% in Southwest Sumba (combined: 31.8%). Among pneumococcal isolates identified, the most common VT were 6B (16.4%), 19F (15.8%), and 3 (4.6%) in Gunungkidul (N = 323) and 6B (17.6%), 19F (11.0%), and 23F (9.3%) in Southwest Sumba (N = 784). Factors associated with pneumococcal carriage were age (1-2 years adjusted odds ratio (aOR) 1.9, 95% CI 1.4-2.5; 3-4 years aOR 1.5, 95% CI 1.1-2.1; reference <1 year), other children <5 years old in the household (aOR 1.5, 95% CI 1.1-2.0), and presence of ≥1 respiratory illness symptom (aOR 1.8, 95% CI 1.4-2.2). Overall, 61.5% of the pneumococcal isolates were non-susceptible to ≥1 antibiotic class and 13.2% were multi-drug non-susceptible (MDNS) (non-susceptible to ≥3 classes of antibiotics). Among 602 VT isolates, 73.9% were non-susceptible and 19.9% were MDNS. These findings are critical to establish a pre-PCV13 carriage prevalence and demonstrate the complexity in evaluating the impact of PCV13 introduction in Indonesia given the wide variability in the carriage prevalence as shown by the two study sites.

Nasopharyngeal colonization by Streptococcus pneumoniae in children and adults before the introduction of the 10-valent conjugate vaccine, Paraguay.

Streptococcus pneumoniae is a cause of invasive diseases such as pneumonia, meningitis, and other serious infections among children and adults in Paraguay. This study was conducted to establish S. pneumoniae baseline prevalence, serotype distribution, and antibiotic resistance patterns in healthy children aged 2 to 59 months and adults ≥60 years of age prior to the introduction of PCV10 in the national childhood immunization program in Paraguay. Between April and July 2012, a total of 1444 nasopharyngeal swabs were collected, 718 from children aged 2 to 59 months and 726 from adults ≥60 years of age. The pneumococcal isolation, serotyping, and antibiotic susceptibility testing were performed using standard tests. Pneumococcal colonization prevalence was 34.1% (245/718) in children and 3.3% (24/726) in adults. The most frequent pneumococcal vaccine-types (VT) detected in the children were 6B (42/245), 19F (32/245), 14 (17/245), and 23F (20/245). Carriage prevalence with PCV10 serotypes was 50.6% (124/245) and PCV13 was 59.5% (146/245). Among colonized adults, prevalence of PCV10 and PCV13 serotypes were 29.1% (7/24) and 41.6% (10/24), respectively. Colonized children were more likely to share a bedroom, have a history of respiratory infection or pneumococcal infection compared to non-colonized children. no associations were found in adults. However, no significant associations were found in children and neither in adults. Vaccine-type pneumococcal colonization was highly prevalent in children and rare in adults in Paraguay prior to vaccine introduction, supporting the introduction of PCV10 in the country in 2012. These data will be useful to evaluate the impact of PCV introduction in the country.

Histopathology Is Key to Interpreting Multiplex Molecular Test Results From Postmortem Minimally Invasive Tissue Samples.

BACKGROUND: Minimally invasive tissue sampling (MITS) is an alternative to complete autopsy for determining causes of death. Multiplex molecular testing performed on MITS specimens poses challenges of interpretation, due to high sensitivity and indiscriminate detection of pathogenic, commensal, or contaminating microorganisms. METHODS: MITS was performed on 20 deceased children with respiratory illness, at 10 timepoints up to 88 hours postmortem. Samples were evaluated by multiplex molecular testing on fresh tissues by TaqMan® Array Card (TAC) and by histopathology, special stains, immunohistochemistry (IHC), and molecular testing (PCR) on formalin-fixed, paraffin-embedded (FFPE) tissues. Results were correlated to determine overall pathologic and etiologic diagnoses and to guide interpretation of TAC results. RESULTS: MITS specimens collected up to 3 days postmortem were adequate for histopathologic evaluation and testing. Seven different etiologic agents were detected by TAC in 10 cases. Three cases had etiologic agents detected by FFPE or other methods and not TAC; 2 were agents not present on TAC, and 2 were streptococci that may have been species other than those present on TAC. Result agreement was 43% for TAC and IHC or PCR, and 69% for IHC and PCR. Extraneous TAC results were common, especially when aspiration was present. CONCLUSIONS: TAC can be performed on MITS up to 3 days after death with refrigeration and provides a sensitive method for detection of pathogens but requires careful interpretation in the context of clinicoepidemiologic and histopathologic findings. Interpretation of all diagnostic tests in aggregate to establish overall case diagnoses maximizes the utility of TAC in MITS.

Prevalence, serotype and antibiotic susceptibility of Group B Streptococcus isolated from pregnant women in Jakarta, Indonesia.

Group B Streptococcus (GBS) is a bacterial pathogen which is a leading cause of neonatal infection. Currently, there are limited GBS data available from the Indonesian population. In this study, GBS colonization, serotype distribution and antimicrobial susceptibility profile of isolates were investigated among pregnant women in Jakarta, Indonesia. Demographics data, clinical characteristics and vaginal swabs were collected from 177 pregnant women (mean aged: 28.7 years old) at 29-40 weeks of gestation. Bacterial culture identification tests and latex agglutination were performed for GBS. Serotyping was done by conventional multiplex PCR and antibiotic susceptibility testing by broth microdilution. GBS colonization was found in 53 (30%) pregnant women. Serotype II was the most common serotype (30%) followed by serotype III (23%), Ia and IV (13% each), VI (8%), Ib and V (6% each), and one non-typeable strain. All isolates were susceptible to vancomycin, penicillin, ampicillin, cefotaxime, daptomycin and linezolid. The majority of GBS were resistant to tetracycline (89%) followed by clindamycin (21%), erythromycin (19%), and levofloxacin (6%). The serotype III was more resistant to erythromycin, clindamycin, and levofloxacin and these isolates were more likely to be multidrug resistant (6 out of 10) compared to other serotypes. This report provides demographics of GBS colonization and isolate characterization in pregnant women in Indonesia. The results may facilitate preventive strategies to reduce neonatal GBS infection and improve its treatment.

Nonpneumococcal Strains Recently Recovered from Carriage Specimens and Expressing Capsular Serotypes Highly Related or Identical to Pneumococcal Serotypes 2, 4, 9A, 13, and 23A.

The polysaccharide capsule is a key virulence factor of Streptococcus pneumoniae There are numerous epidemiologically important pneumococcal capsular serotypes, and recent findings have demonstrated that several of them are commonly found among nonpathogenic commensal species. Here, we describe 9 nonpneumococcal strains carrying close homologs of pneumococcal capsular biosynthetic (cps) loci that were discovered during recent pneumococcal carriage studies of adults in the United States and Kenya. Two distinct Streptococcus infantis strains cross-reactive with pneumococcal serotype 4 and carrying cps4-like capsular biosynthetic (cps) loci were recovered. Opsonophagocytic killing assays employing rabbit antisera raised against S. infantis US67cps4 revealed serotype 4-specific killing of both pneumococcal and nonpneumococcal strains. An S. infantis strain and two Streptococcus oralis strains, all carrying cps9A-like loci, were cross-reactive with pneumococcal serogroup 9 strains in immunodiffusion assays. Antiserum raised against S. infantis US64cps9A specifically promoted killing of serotype 9A and 9V pneumococcal strains as well as S. oralis serotype 9A strains. Serotype-specific PCR of oropharyngeal specimens from a recent adult carriage study in the United States indicated that such nonpneumococcal strains were much more common in this population than serotype 4 and serogroup 9 pneumococci. We also describe S. oralis and S. infantis strains expressing serotypes identical or highly related to serotypes 2, 13, and 23A. This study has expanded the known overlap of pneumococcal capsular serotypes with related commensal species. The frequent occurrence of nonpneumococcal strains in the upper respiratory tract that share vaccine and nonvaccine capsular serotypes with pneumococci could affect population immunity to circulating pneumococcal strains.IMPORTANCE The distributions and frequencies of individual pneumococcal capsular serotypes among nonpneumococcal strains in the upper respiratory tract are unknown and potentially affect pneumococcal serotype distributions among the population and immunity to circulating pneumococcal strains. Repeated demonstration that these nonpneumococcal strains expressing so-called pneumococcal serotypes are readily recovered from current carriage specimens is likely to be relevant to pneumococcal epidemiology, niche biology, and even to potential strategies of employing commensal live vaccines. Here, we describe multiple distinct nonpneumococcal counterparts for each of the pneumococcal conjugate vaccine (PCV) serotypes 4 and 9V. Additional data from contemporary commensal isolates expressing serotypes 2, 13, and 23A further demonstrate the ubiquity of such strains. Increased focus upon this serological overlap between S. pneumoniae and its close relatives may eventually prove that most, or possibly all, pneumococcal serotypes have counterparts expressed by the common upper respiratory tract commensal species Streptococcus mitis, Streptococcus oralis, and Streptococcus infantis.

Effect of 10-Valent Pneumococcal Conjugate Vaccine on Streptococcus pneumoniae Nasopharyngeal Carriage Among Children Less Than 5 Years Old: 3 Years Post-10-Valent Pneumococcal Conjugate Vaccine Introduction in Mozambique.

BACKGROUND: Mozambique introduced 10-valent pneumococcal conjugate vaccine (PCV10) in 2013 with doses at ages 2, 3, and 4 months and no catch-up or booster dose. We evaluated PCV10 impact on the carriage of vaccine-type (VT), non-VT, and antimicrobial non-susceptible pneumococci 3 years after introduction. METHODS: We conducted cross-sectional carriage surveys among HIV-infected and HIV-uninfected children aged 6 weeks to 59 months: 1 pre-PCV10 (2012-2013 [Baseline]) and 2 post-PCV10 introductions (2014-2015 [Post1] and 2015-2016 [Post2]). Pneumococci isolated from nasopharyngeal swabs underwent Quellung serotyping and antimicrobial susceptibility testing. Non-susceptible isolates (intermediate or resistant) were defined using Clinical and Laboratory Standards Institute 2018 breakpoints. We used log-binomial regression to estimate changes in the pneumococcal carriage between survey periods. We compared proportions of non-susceptible pneumococci between Baseline and Post2. RESULTS: We enrolled 720 children at Baseline, 911 at Post1, and 1208 at Post2. Baseline VT carriage was similar for HIV-uninfected (36.0%, 110/306) and HIV-infected children (34.8%, 144/414). VT carriage was 36% (95% confidence interval [CI]: 19%-49%) and 27% (95% CI: 11%-41%) lower in Post1 vs baseline among HIV-uninfected and HIV-infected children, respectively. VT carriage prevalence declined in Post2 vs Post1 for HIV-uninfected but remained stable for HIV-infected children. VT carriage prevalence 3 years after PCV10 introduction was 14.5% in HIV-uninfected and 21.0% in HIV-infected children. Pneumococcal isolates non-susceptible to penicillin declined from 66.0% to 56.2% (P= .0281) among HIV-infected children. CONCLUSIONS: VT and antimicrobial non-susceptible pneumococci carriage dropped after PCV10 introduction, especially in HIV-uninfected children. However, VT carriage remained common, indicating ongoing VT pneumococci transmission.

Limited Added Value of Oropharyngeal Swabs for Detecting Pneumococcal Carriage in Adults.

We compared pneumococcal isolation rates and evaluated the benefit of using oropharyngeal (OP) specimens in addition to nasopharyngeal (NP) specimens collected from adults in rural Kenya. Of 846 adults, 52.1% were colonized; pneumococci were detected from both NP and OP specimens in 23.5%, NP only in 22.9%, and OP only in 5.7%. Ten-valent pneumococcal conjugate vaccine strains were detected from both NP and OP in 3.4%, NP only in 4.1%, and OP only in 0.7%. Inclusion of OP swabs increased carriage detection by 5.7%; however, the added cost of collecting and processing OP specimens may justify exclusion from future carriage studies among adults.

Dried blood spots for Streptococcus pneumoniae and Haemophilus influenzae detection and serotyping among children < 5 years old in rural Mozambique.

BACKGROUND: Dried blood spots (DBS) have been proposed as potentially tool for detecting invasive bacterial diseases. METHODS: We evaluated the use of DBS for S. pneumoniae and H. influenzae detection among children in Mozambique. Blood for DBS and nasopharyngeal (NP) swabs were collected from children with pneumonia and healthy aged < 5 years. Bacterial detection and serotyping were performed by quantitative PCR (qPCR) (NP and DBS; lytA gene for pneumococcus and hpd for H. influenzae) and culture (NP). Combined detection rates were compared between children with pneumonia and healthy. RESULTS: Of 325 children enrolled, 205 had pneumonia and 120 were healthy. Pneumococci were detected in DBS from 20.5 and 64.2% of children with pneumonia and healthy, respectively; NP specimens were positive for pneumococcus in 80.0 and 80.8%, respectively. H. influenzae was detected in DBS from 22.9% of children with pneumonia and 59.2% of healthy; 81.4 and 81.5% of NP specimens were positive for H. influenzae, respectively. CONCLUSION: DBS detected pneumococcal and H. influenzae DNA in children with pneumonia and healthy. Healthy children were often DBS positive for both bacteria, suggesting that qPCR of DBS specimens does not differentiate disease from colonization and is therefore not a useful diagnostic tool for children.

Invasive disease potential of Streptococcus pneumoniae serotypes before and after 10-valent pneumococcal conjugate vaccine introduction in a rural area, southern Mozambique.

BACKGROUND: Invasive pneumococcal disease (IPD) is a significant cause of morbidity and mortality among children worldwide. In April 2013, Mozambique introduced 10-valent PCV (PCV10) into the National Expanded Program on immunization using a three-dose schedule at 2, 3, and 4 months of age. We aimed to evaluate the invasive disease potential of pneumococcal serotypes among children in our region before and after PCV10 introduction. METHODS: We used data from ongoing population-based surveillance for IPD and cross-sectional pneumococcal carriage surveys among children aged <5 years in Manhiҫa, Mozambique. To determine the invasive disease potential for each serotype pre- and post-PCV10 introduction, odds ratios (OR) and 95% confidence intervals (95% CI) were calculated comparing serotype-specific prevalence in IPD and in carriage. For each serotype, OR and 95% CI > 1 indicated high invasive disease potential and OR and 95% CI < 1 indicated low invasive disease potential. RESULTS: In the pre-PCV10 period, 524 pneumococcal isolates were obtained from 411 colonized children and IPD cases were detected in 40 children. In the post-PCV10 period, 540 pneumococcal isolates were obtained from 507 colonized children and IPD cases were detected in 30 children. The most prevalent serotypes causing IPD pre-PCV10 were 6A (17.5%), 6B (15.0%), 14 (12.5%), 23F (10.0%) and 19F (7.5%), and post-PCV10 were 6A (36.7%), 13 (10%), 1 (10.0%), 6B (6.7%) and 19A (6.7%). Serotypes associated with high invasive disease potential pre-PCV10 included 1 (OR:22.3 [95% CI 2.0; 251.2]), 6B (OR:3.1 [95% CI 1.2; 8.1]), 14 (OR: 3.4 [95% CI 1.2; 9.8]) and post-PCV10 included serotype 6A (OR:6.1[95% CI 2.7; 13.5]). CONCLUSION: The number of serotypes with high invasive disease potential decreased after PCV10 introduction. Serotype 6A, which is not included in PCV10, was the most common cause of IPD throughout the study and showed a high invasive potential in the post-PCV10 period.

How to avoid cross contamination during handling resin composites with spatulas / Como evitar a contaminação cruzada durante a manipulação de resinas compostas com espátulas

Incremental technique for resin composite restorations requires multiple physical contacts between spatulas, tooth cavities and the restorative material recipient. Thus, the decontamination of the tip's of spatula by chemical agents between each resin composite increment placement is important to reduce chances of potential cross-contamination. Objectives: To evaluate the efficacy of different solutions for decontamination of tips of spatula used in restorative procedures and to establish a decontamination standard protocol. Material and Methods: Spatulas were sterilized in autoclave at 127°C for 20 minutes and then contaminated with: 1) a suspension of half 1.0 MacFarland scale turbidity of different microorganisms, 2) the pool of equal amounts of these microorganisms; except for the control group. Decontamination techniques consisted of rubbing the tip of the spatulas (1 to 5 consecutive times) using a 2ml 70% ethanol or 2% glutaraldehyde embedded gauze. After decontamination, spatulas were immersed in thioglycolate broth and incubated for 48 hours at 37°C. Broth with visible microbial detection was submitted to bacterial identification by Gram stain. Results: Low uniformity of rubbings number was observed to eliminate different microorganisms due to different tested disinfectant agents. Four or five rubbings were needed to decontamination of the tested microorganisms using 70% ethanol. Three rubbings using 2% glutaraldehyde were able to eliminate tested microorganisms. Conclusion: The results demonstrated that 70% ethanol by friction, counting four or five rubbings, was effective to decontaminate spatula's tip.

A técnica incremental para restaurações de resina composta requer vários contatos físicos entre as espátulas, o preparo cavitário e a embalagem do material restaurador. Assim, a descontaminação da ponta da espátula por meio de agentes químicos entre cada inserção de um novo incremento de resina composta é importante para reduzir as chances de uma potencial de contaminação cruzada. Objetivos: Avaliar a eficácia de diferentes soluções para descontaminação das pontas das espátulas utilizadas em procedimentos restauradores e estabelecer um protocolo padrão de descontaminação. Material e Métodos: espátulas foram esterilizadas em autoclave a 127 °C durante 20 minutos e, em seguida, contaminadas com: 1) uma suspensão de diferentes microorganismos com turbidez equivalente ao padrão 1,0 da escala de McFarland, 2) pool de quantidades iguais destes microrganismos; exceto para o grupo de controle. As técnicas de descontaminação consistiram em esfregar a ponta das espátulas (1 a 5 vezes consecutivas) utilizando 2 ml de álcool 70% ou 2% de glutaraldeído embebidos em gazes. Após a descontaminação, as espátulas foram imersas em caldo de tioglicolato e incubadas durante 48 horas a 37 °C. O caldo com visível detecção microbiana foi submetido à identificação bacteriana pela coloração de Gram. Resultados: Baixa uniformidade do esfregaço foi observada para a eliminação de diferentes microrganismos, devido aos diferentes agentes desinfetantes testados. Quatro ou cinco esfregaços foram necessários para a descontaminação dos microrganismos testados usando álcool 70%. Três esfregaços na espátula usando glutaraldeído a 2% foram capazes de eliminar os microrganismos testados. Conclusão: Os resultados demonstraram que o álcool 70% por fricção, contando quatro ou cinco esfregaços na espátula com gazes embebida nas soluções desinfetantes, foi eficaz para descontaminar a ponta da espátula.

Direct effect of 10-valent conjugate pneumococcal vaccination on pneumococcal carriage in children Brazil.

BACKGROUND: 10-valent conjugate pneumococcal vaccine/PCV10 was introduced in the Brazilian National Immunization Program along the year of 2010. We assessed the direct effectiveness of PCV10 vaccination in preventing nasopharyngeal/NP pneumococcal carriage in infants. METHODS: A cross-sectional population-based household survey was conducted in Goiania Brazil, from December/2010-February/2011 targeting children aged 7-11 m and 15-18 m. Participants were selected using a systematic sampling. NP swabs, demographic data, and vaccination status were collected from 1,287 children during home visits. Main outcome and exposure of interest were PCV10 vaccine-type carriage and dosing schedules (3p+0, 2p+0, and one catch-up dose), respectively. Pneumococcal carriage was defined by a positive culture and serotyping was performed by Quellung reaction. Rate ratio/RR was calculated as the ratio between the prevalence of vaccine-types carriage in children exposed to different schedules and unvaccinated for PCV10. Adjusted RR was estimated using Poisson regression. PCV10 effectiveness/VE on vaccine-type carriage was calculated as 1-RR*100. RESULTS: The prevalence of pneumococcal carriage was 41.0% (95%CI: 38.4-43.7). Serotypes covered by PCV10 and PCV13 were 35.2% and 53.0%, respectively. Vaccine serotypes 6B (11.6%), 23F (7.8%), 14 (6.8%), and 19F (6.6%) were the most frequently observed. After adjusted for confounders, children who had received 2p+0 or 3p+0 dosing schedule presented a significant reduction in pneumococcal vaccine-type carriage, with PCV10 VE equal to 35.9% (95%CI: 4.2-57.1; p = 0.030) and 44.0% (95%CI: 14.-63.5; p = 0.008), respectively, when compared with unvaccinated children. For children who received one catch-up dose, no significant VE was detected (p = 0.905). CONCLUSION: PCV10 was associated with high protection against vaccine-type carriage with 2p+0 and 3p+0 doses for children vaccinated before the second semester of life. The continuous evaluation of carriage serotypes distribution is likely to be useful for evaluating the long-term effectiveness and impact of pneumococcal vaccination on serotypes reduction.

Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract.

We performed culture-based and PCR-based tests for pneumococcal identification and serotyping from carriage specimens collected in rural and urban Kenya. Nasopharyngeal specimens from 237 healthy children <5 years old (C-NPs) and combined nasopharyngeal/oropharyngeal specimens from 158 adults (A-NP/OPs, 118 HIV-positive) were assessed using pneumococcal isolation (following broth culture enrichment) with Quellung-based serotyping, real-time lytA-PCR, and conventional multiplexed PCR-serotyping (cmPCR). Culture-based testing from C-NPs, HIV-positive A-NP/OPs, and HIV-negative A-NP/OPs revealed 85.2%, 40.7%, and 12.5% pneumococcal carriage, respectively. In contrast, cmPCR serotypes were found in 93.2%, 98.3%, and 95.0% of these sets, respectively. Two of 16 lytA-negative C-NPs and 26 of 28 lytA-negative A-NP/OPs were cmPCR-positive for 1-10 serotypes (sts) or serogroups (sgs). A-NP/OPs averaged 5.5 cmPCR serotypes/serogroups (5.2 in HIV-positive, 7.1 in HIV-negative) and C-NPs averaged 1.5 cmPCR serotypes/serogroups. cmPCR serotypes/serogroups from lytA-negative A-NP/OPs included st2, st4, sg7F/7A, sg9N/9L, st10A, sg10F/10C/33C, st13, st17F, sg18C/18A/18B/18F, sg22F/22A, and st39. Nine strains of three non-pneumococcal species (S. oralis, S. mitis, and S. parasanguinis) (7 from A-OP, 1 from both A-NP and A-OP, and 1 from C-NP) were each cmPCR-positive for one of 7 serotypes/serogroups (st5, st13, sg15A/15F, sg10F/10C/33C, sg33F/33A/37, sg18C/18A/18B/18F, sg12F/12A/12B/ 44/46) with amplicons revealing 83.6-99.7% sequence identity to pneumococcal references. In total, 150 cmPCR amplicons from carriage specimens were sequenced, including 25 from lytA-negative specimens. Amplicon sequences derived from specimens yielding a pneumococcal isolate with the corresponding serotype were identical or highly conserved (>98.7%) with the reference cmPCR amplicon for the st, while cmPCR amplicons from lytA-negative specimens were generally more divergent. Separate testing of 56 A-OPs and 56 A-NPs revealed that ∼94% of the positive cmPCR results from A-NP/OPs were from OP microbiota. In contrast, A-NPs yielded >2-fold more pneumococcal isolates than A-OPs. Verified and suspected non-pneumococcal cmPCR serotypes/serogroups appeared to be relatively rare in C-NPs and A-NPs compared to A-OPs. Our findings indicate that non-pneumococcal species can confound serotype-specific PCR and other sequence-based assays due to evolutionarily conserved genes most likely involved in biosynthesis of surface polysaccharide structures.

Sequential triplex real-time PCR assay for detecting 21 pneumococcal capsular serotypes that account for a high global disease burden.

We developed and validated a real-time PCR assay consisting of 7 triplexed reactions to identify 11 individual serotypes plus 10 small serogroups representing the majority of disease-causing isolates of Streptococcus pneumoniae. This assay targets the 13 serotypes included within the 13-valent conjugate vaccine and 8 additional key serotypes or serogroups. Advantages over other serotyping assays are described. The assay will be expanded to 40 serotypes/serogroups. We will provide periodic updates at our protocol website.

Serotype and genotype distributions of pneumococcal carriage isolates recovered from Brazilian children attending day-care centres.

Pneumococcal nasopharyngeal carriage isolates recovered from Brazilian children attending day-care centres in 2005 were assessed for serotype, genotype and penicillin susceptibility phenotype. As 124 of the 253 isolates (49 %) were characterized previously with respect to serotype and penicillin susceptibility, the primary objectives were to examine clonal associations and penicillin susceptibility within major serotypes and to assess the suitability of conventional multiplex PCR for deducing carriage serotypes within this population. Using a combination of PCR-based serotyping and the Quellung reaction, serotypes were identified for 81 % (205/253) of the isolates, with serogroups or types 14, 6, 23F, 19F and 18 being predominant. Included within the 205 isolates successfully serotyped by PCR were 28 isolates that had become non-viable. Forty-eight isolates were non-typable using both the PCR method and the Quellung reaction. Penicillin non-susceptibility was observed within 16 of the 18 multilocus sequence types detected. Thus, this study provides further evidence from a diverse collection of pneumococcal clones that PCR-based serotype deduction is useful for providing supportive evidence for pneumococcal conjugate vaccine implementation.

Selection of family 1 PspA molecules capable of inducing broad-ranging cross-reactivity by complement deposition and opsonophagocytosis by murine peritoneal cells.

PspA is one of the most well studied pneumococcal proteins and a promising candidate for a future protein-based anti-pneumococcal vaccine. Nevertheless, its structural and serological variability suggests the inclusion of more than one PspA molecule in order to broaden protection. Since different PspAs exhibit variable levels of cross-reactivity, the selection of the protein combination with the highest coverage potential is an essential step for PspA-based vaccine development. This work investigated the level of cross-reactivity within family 1 PspAs, and established a complement based antibody mediated opsonophagocytic assay for measuring the level of cross-protection. Among a panel of ten family 1 PspA molecules, two of them, one belonging to clade 1 and another from clade 2, induced antibodies capable of enhancing complement deposition and mediating the phagocytic killing by mouse peritoneal macrophages of all pneumococci bearing PspA family 1 strains tested, regardless of their serotype. Therefore, we suggest the inclusion of either one in a PspA-based vaccine, as a representative of family 1. Furthermore, our results suggest that opsonophagocytosis by mouse peritoneal cells can be an efficient means of evaluating the induction of protective immune responses in mice across a large number of strains.

Selection of family 1 PspA molecules capable of inducing broad-ranging cross-reactivity by complement deposition and opsonophagocytosis by murine peritoneal cells

PspA is one of the most well studied pneumococcal proteins and a promising candidate for a future protein-based anti-pneumococcal vaccine. Nevertheless, its structural and serological variability suggests the inclusion of more than one PspA molecule in order to broaden protection. Since different PspAs exhibit variable levels of cross-reactivity, the selection of the protein combination with the highest coverage potential is an essential step for PspA-based vaccine development. This work investigated the level of cross-reactivity within family 1 PspAs, and established a complement based antibody mediated opsonophagocytic assay for measuring the level of cross-protection. Among a panel of ten family 1 PspA molecules, two of them, one belonging to clade 1 and another from clade 2, induced antibodies capable of enhancing complement deposition and mediating the phagocytic killing by mouse peritoneal macrophages of all pneumococci bearing PspA family 1 strains tested, regardless of their serotype. Therefore, we suggest the inclusion of either one in a PspA-based vaccine, as a representative of family 1. Furthermore, our results suggest that opsonophagocytosis by mouse peritoneal cells can be an efficient means of evaluating the induction of protective immune responses in mice across a large number of strains.

Pre- and post-conjugate vaccine epidemiology of pneumococcal serotype 6C invasive disease and carriage within Navajo and White Mountain Apache communities.

BACKGROUND: A second-generation 13-valent pneumococcal conjugate vaccine, PCV13, was recently licensed. Although PCV13 includes serotype 6A, the usefulness of that antigen may be limited by the emergence of a new serotype, 6C, which was identified among isolates initially characterized (Quellung reaction) as serotype 6A. The epidemiology of serotype 6C prior to and after 7-valent PCV (PCV7) introduction is incompletely understood. METHODS: We analyzed conventionally serotyped 6A (CS6A) pneumococci from invasive disease case patients of all ages and carriage isolates from children and adults obtained in population-based studies among Navajo and White Mountain Apache communities during 1994-2009. Samples were tested by triplex polymerase chain reaction to resolve serotypes 6C and 6A. RESULTS: A total of 74 invasive CS6A episodes occurred. All were retyped by polymerase chain reaction; 40 (54.1%) were serotype 6C. The mean annual incidence of serotype 6C invasive disease was 0.3 (95% confidence interval, 0.03-0.9), 0.7 (95% confidence interval, 0.2-1.3), and 1.5 (95% confidence interval, 1.0-2.1) cases per 100,000 population in the years prior to the PCV7 efficacy trial, during the time the PCV7 trial was conducted, and following PCV7 introduction and routine use, respectively (P = .01). In the routine vaccination era, 76% of invasive CS6As were serotype 6C; nearly all cases occurred in adults. The proportion of serotype 6C among CS6A carriage isolates increased from 42% to 61% to 94% in the prevaccine, early vaccine, and routine vaccination eras, respectively. CONCLUSION: In the PCV7 routine use era, virtually all serogroup 6 invasive pneumococcal disease and carriage strains among Navajo and White Mountain Apache communities are 6C. Monitoring and evaluation of this and other emerging serotypes among invasive disease and carriage isolates is warranted.

Association of 2009 pandemic influenza A (H1N1) infection and increased hospitalization with parapneumonic empyema in children in Utah.

BACKGROUND: During previous influenza pandemics, many deaths were associated with secondary bacterial infection. In April 2009, a previously unknown 2009 influenza A virus (2009 H1N1) emerged, causing a global influenza pandemic. We examined the relationship between circulating 2009 H1N1 and the occurrence of secondary bacterial parapneumonic empyema in children. METHODS: Children hospitalized with parapneumonic empyema from August 2004 to July 2009, including a period when the 2009 H1N1 circulated in Utah, were identified using International Classification of Diseases, Ninth Revision codes. We compared the average number of children diagnosed with influenza A and the number of admissions for empyema per month for the previous 4 seasons to rates of empyema during the 2009 H1N1 outbreak. We identified causative bacteria using culture and polymerase chain reaction (PCR). RESULTS: We observed an increase in hospitalization of children with pneumonia complicated by empyema during a severe outbreak of 2009 H1N1 during the spring and summer of 2009, compared with historical data for the previous 4 seasons. Streptococcus pneumoniae and Streptococcus pyogenes were the predominant bacteria identified. CONCLUSIONS: Similar to previous pandemics, secondary bacterial infection with S. pneumoniae and S. pyogenes were associated with the 2009 H1N1 outbreak. There is an urgent need to better understand bacterial complications of pandemic influenza. In the interim, influenza vaccines, antiviral agents, and pneumococcal vaccines should be used to prevent cases of secondary bacterial pneumonia whenever possible.