Quellung Method Research Articles

Optimization of a high-throughput nanofluidic real-time PCR to detect and quantify of 15 bacterial species and 92 Streptococcus pneumoniae serotypes

Sensitive tools for detecting concurrent colonizing pneumococcal serotypes are needed for detailed evaluation of the direct and indirect impact of routine pneumococcal conjugate vaccine (PCV) immunization. A high-throughput quantitative nanofluidic real-time PCR (Standard BioTools ‘Fluidigm’) reaction-set was developed to detect and quantify 92 pneumococcal serotypes in archived clinical samples. Nasopharyngeal swabs collected in 2009–2011 from South African children ≤ 5 years-old, previously serotyped with standard culture-based methods were used for comparison. The reaction-set within the ‘Fluidigm’ effectively amplified all targets with high efficiency (90–110%), reproducibility (R2 ≥ 0.98), and at low limit-of-detection (< 102 CFU/ml). A blind analysis of 1 973 nasopharyngeal swab samples showed diagnostic sensitivity > 80% and specificity > 95% compared with the referent standard, culture based Quellung method. The qPCR method was able to serotype pneumococcal types with good discrimination compared with Quellung (ROC-AUC: > 0.73). The high-throughput nanofluidic real-time PCR method simultaneously detects 57 individual serotypes, and 35 serotypes within 16 serogroups in 96 samples (including controls), within a single qPCR run. This method can be used to evaluate the impact of current PCV formulations on vaccine-serotype and non-vaccine-serotype colonization, including detection of multiple concurrently colonizing serotypes. Our qPCR method can allow for monitoring of serotype-specific bacterial load, as well as emergence or ongoing transmission of minor or co-colonizing serotypes that may have invasive disease potential.

High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides

Current real-time high-throughput Polymerase Chain Reaction (qPCR) methods do not distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009–2011), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92–101%), had low variation between replicates (R2 > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/mL of sample. There was high concordance (Kappa = 0.73–0.92); sensitivity (85–100%) and specificity (96–100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.

Trend change of nasopharyngeal colonization with Streptococcus pneumoniae and non-typeable Haemophilus influenzae in children attending daycare centres: nationwide population-based study, South Korea 2014 and 2019

Nasopharyngeal (NP) colonization with Streptococcus pneumoniae and non-typeable Haemophilus influenzae (NTHi) is common in children, and may evolve as the source of invasive infections. In Korea, the pneumococcal conjugate vaccines (PCVs) were introduced >10 years ago, enabling the authors to study the effect of the vaccine in preventing carriage. NP swabs were taken and a household survey was conducted at daycare centres located in different regions of Korea in 2014 and 2019. Pneumococcal serotypes were identified using the Quellung method and sequencing. NTHi were identified based on pilA and bexA genes. In total, 1460 NP swabs were obtained with pneumococcal carriage rates of 36.4-42.1% and NTHi carriage rates of 36.5-26.7%. Among children carrying pneumococci, a significant increase was seen in serotype 23A between 2014 and 2019 (from 12.6% to 22.0%; P=0.005). Children who had received PCV were at lower risk of vaccine-type carriage (2.9% vs 0.8%; P=0.005). Between 2014 and 2019, the proportion of children carrying serotype 23A increased significantly, while the carriage rate of NTHi decreased. Continuous surveillance is needed to assess the long-term effects of the PCVs on carriage dynamics of pneumococcus and NTHi.

Nasopharyngeal colonization of otopathogens in South Indian children with acute otitis media – A case control pilot study

BackgroundAcute otitis media (AOM) is an inflammatory disease of the middle ear causing significant morbidity in early childhood. A pilot study was undertaken to identify the role of various risk factors South Indian children with AOM, especially the role of nasopharyngeal otopathogens. MethodologyA prospective case control pilot study was conducted in children aged below six years, presenting to a single tertiary care from 2018 to 2019. Fifty cases with AOM and 45 age and gender matched controls were recruited. Two nasopharyngeal swabs were collected, one was processed for bacterial culture. The other swab was processed according to the CDC recommended broth enrichment method to identify carriage of S. pneumoniae. Subsequent serotyping was done by Quellung method and conventional sequential multiplex PCR. ResultOtalgia was the major presentation seen in 92% of the children with AOM. None of the clinical and demographic characteristics were found to be statistically significant between the cases and controls. The most common otopathogen was S. pneumoniae (55%) followed by H. influenza (29%). The common S. pneumoniae serotypes encountered were 11A and 19F.Nasopharyngeal colonization with S. pneumoniae [OR 6.57, p < 0.003] and H. influenzae [OR14.18, p < 0.003] were significant risk factors for AOM in children. The risk increased with co-colonization (OR 13.89,p < 0.003). ConclusionThis study strengthens the significant association between nasopharyngeal colonization of otopathogens and AOM as a risk factor that is enhanced by co-colonization.S. pneumoniae was the main otopathogen in this population, serotypes 11A and 19F being the most common.

Nasopharyngeal carriage of Streptococcus pneumoniae serotypes among healthy children in North India

Background: Streptococcus pneumonia (SP) is a major cause of morbidity and mortality among children worldwide. India introduced 13-valent pneumococcal conjugate vaccine (PCV-13) in 2017 in national immunization program in a three dose schedule at 6 weeks, 10 weeks and 9 months. The current study was conducted to isolate SP from nasopharyngeal (NP) swabs of healthy children and assess changes in serotypes among PCV vaccinated and unvaccinated healthy children. Methods and materials: Cross-sectional study was conducted in Lucknow District, Uttar Pradesh, India from July–August 2019. Recruitment was done from vaccination clinics of four public tertiary care hospitals. Children (2–59 months) who were permanent residents and had no clear illness/hospitalization in past one month were recruited. Single NP specimen was collected. After specimen collection, bacterial culture was done using 5% sheep agar blood plate containing gentamicin. Pneumococcal isolates were identified by optochin sensitivity and bile solubility. Serotyping was done using Quellung Method. Results: Of 300 children, 56.7% (170/300) were males and 52.0%, (156/300) were 2–11 months of age. Overall SP colonization rate was 37.6% (113/300). Vaccine serotypes isolated were 18 C, 19 A, 19 F, 23F, 3, 4, 6A, 6B, 9V. Among 60% (181/300) PCV vaccinated, SP positivity was 37.5% (68/181) and vaccine serotype were 39.7% (27/68). Among 40% (119/300) non PCV vaccinated children, 37.8% (47/119) had NP colonization positive and PCV-13 serotypes were 37.7% (17/47). Non Vaccine serotypes were 10A, 15A, 15B, 15C, 21, 34, 35B, 22F and similar in vaccinated and unvaccinated children. Conclusion: About 4 in 10 health children under 5 years of age had NP colonization with SP and around 40% serotypes were covered by PCV13. Our preliminary data suggests that SP colonization rate and serotype isolates were similar in vaccinated and unvaccinated children, however, further work is needed.

Major Adverse Cardiovascular Events During Invasive Pneumococcal Disease Are Serotype Dependent.

Up to 30% of patients admitted to hospitals with invasive pneumococcal disease (IPD) experience major adverse cardiovascular event (MACE) including new/worsening heart failure, new/worsening arrhythmia, and/or myocardial infarction. Streptococcus pneumoniae (Spn) is the most frequently isolated bacterial pathogen among community-acquired pneumonia (CAP) patients and the only etiological agent linked independently to MACE. Nevertheless, no clinical data exist identifying which serotypes of Spn are principally responsible for MACE. This was an observational multicenter retrospective study conducted through the Public Health Secretary of Bogotá, Colombia. We included patients with a confirmed clinical diagnosis of IPD with record of pneumococcal serotyping and clinical information between 2012 and 2019. Spn were serotyped using the quellung method by the National Center of Microbiology. MACE were determined by a retrospective chart review. The prevalence of MACE was 23% (71/310) in IPD patients and 28% (53/181) in patients admitted for CAP. The most prevalent S. pneumoniae serotype identified in our study was the 19A, responsible for the 13% (42/310) of IPD in our cohort, of which 21% (9/42) presented MACE. Serotypes independently associated with MACE in IPD patients were serotype 3 (odds ratio [OR] 1, 48; 95% confidence interval [CI] [1.21-2.27]; P = .013) and serotype 9n (OR 1.29; 95% CI [1.08-2.24]; P = .020). Bacteremia occurred in 87% of patients with MACE. Moreover, serum concentrations of C-reactive protein were elevated in patients with MACE versus in non-MACE patients (mean [standard deviation], 138 [145] vs 73 [106], P = .01). MACE are common during IPD with serotype 3 and 9n independently of frequency.

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.

A risk assessment of pathogenic Streptococcus suis in pork supply chains and markets in Thailand

Annually, hundreds of severe foodborne cases related to Streptococcus suis (S. suis) have been reported in Southeast Asia, including Thailand. This study aimed at quantitatively evaluating the S. suis contaminations in pork and surrounding environment in pork production chains and its contributions to human illnesses. Pig-associated and environmental samples were collected from four pork supply chains in Bangkok, and cross-sectional pork samples were collected from traditional market and supermarkets in eight provinces in Thailand. All samples were enumerated for S. suis serotype 2 using standard plate count method, and bacterial species were confirmed by biochemical tests together with PCR assays and Quellung method. A quantitative microbial risk assessment (QMRA) was performed to evaluate the S. suis serotype 2 illness risk from consuming pork in Thailand. Among four pork supply chains, the mean S. suis prevalence of pig-associated, and environmental samples were 10.9% (54/496), and 5.21% (25/480), respectively. S. suis has the highest prevalence (56.3%) (9/16), and the highest concentration (5.6 log CFU/g) in palatine tonsils. S. suis was isolated from 0.93% (11/1180) of pork samples; none of them was serotype 2. The QMRA model revealed a mean daily probability of S. suis serotype 2 illness as 2.32 × 10−8, corresponding to 502 and 31 annual cases and deaths, respectively. Additionally, the illness risk from consuming raw pork was predicted at 5.47 × 10−6. In conclusion, even the probability of S. suis illnesses from consuming pork was considerably low; however, the illnesses could lead to serious health consequences among susceptible populations.

Serotype-specific effectiveness of 23-valent pneumococcal polysaccharide vaccine against pneumococcal pneumonia in adults aged 65 years or older: a multicentre, prospective, test-negative design study

The serotype-specific effectiveness of 23-valent pneumococcal polysaccharide vaccine (PPV23) against pneumococcal pneumonia has not been established in people aged 65 years or older. We assessed the effectiveness of PPV23 in this population. For this multicentre, prospective study, we enrolled all individuals aged 65 years or older with community-onset pneumonia who visited four study hospitals in Japan between Sept 28, 2011, and Aug 23, 2014. Streptococcus pneumoniae was isolated from sputum and blood samples, and serotyped by the capsular Quellung method. Sputum samples were further tested by PCR assay to identify pneumococcal DNA, and positive samples were examined for 50 serotypes by a nanofluidic real-time PCR assay. Urine samples were tested by a urinary antigen test. Serotype-specific vaccine effectiveness was estimated using the test-negative design. 2621 eligible patients visited the study hospitals, of whom 585 did not have sputum samples available and were excluded from our analysis. 419 (21%) of 2036 patients were positive for pneumococcal infection (232 by sputum culture, 317 by sputum PCR, 197 by urinary antigen test, and 14 by blood culture). 522 (26%) patients were judged to be vaccinated in the analyses. Effectiveness of PPV23 was 27·4% (95% CI 3·2 to 45·6) against all pneumococcal pneumonia, 33·5% (5·6 to 53·1) against PPV23 serotypes, and 2·0% (-78·9 to 46·3) against non-PPV23 serotypes. Although no significant differences between subgroups were seen, higher protection was noted in people younger than 75 years, women, and individuals with lobar pneumonia or health-care-associated pneumonia. PPV23 showed low to moderate effectiveness against vaccine serotype pneumococcal pneumonia in people aged 65 years or older. To improve the current pneumococcal vaccination programme, the variability of PPV23 effectiveness in different groups of older people must be further investigated. Pfizer and Nagasaki University.

Prevalence of Pneumococcal Serotypes in Adults ≥50 Years of Age

Context: Currently, majority of studies on antibiotic resistance and serotype prevalence in Streptococcus pneumoniae are in paediatric population and there is limited epidemiologic data pertaining to elderly Indian population. Aims: This study was undertaken to assess antibiotic resistance pattern and the coverage of the polysaccharide vaccine 23 (PPV23) in the elderly population. Settings and Design: This was a prospective 2-year pneumococcal surveillance study in patients with community-acquired pneumococcal infections of ≥50 years’ age group on fifty isolates of S. pneumoniae. Materialsand Methods: Antibiotic susceptibility by Kirby-Bauer disc diffusion method and minimum inhibitory concentration susceptibility testing by E-test method were performed for various antibiotics. All isolates were serotyped by the capsular Quellung method with commercial antisera. Results: All S. pneumoniae isolates were sensitive to penicillin and ceftriaxone. Non-susceptibility to levofloxacin, co-trimoxazole, erythromycin and clindamycin was noted as 16%, 74%, 10% and 6%, respectively. Prevalence of multidrug resistance in S. pneumoniae was observed as 6%. The most common serotypes observed in our study were 19A (14%), 8 (10%), 19F (8%), 3 (6%) and 9N (6%). PCV7, PCV10, PCV13 and PCV23 coverage was observed as 16%, 24%, 48% and 66%, respectively. The non-vaccine serotypes (NVTs) comprised 30% (n = 15) of the isolates. Conclusions: Our study shows different antibiotic susceptibility patterns of S. pneumoniae as compared to other neighbouring regions in Asia. The modest coverage of S. pneumoniae serotypes by PPV23 vaccine and prevalence of diverse NVTs in adult population make it a challenging task to recommend further changes in the future vaccine.

Prevalence of antimicrobial resistant Streptococcus pneumoniae serotype 11A isolates in Korea, during 2004–2013, due to the increase of multidrug-resistant clone, CC166

Since the introduction of the pneumococcal conjugate vaccine (PCV7) in Korea in 2003, the proportion of non-vaccine serotypes has increased. Among non-vaccine serotypes, serotype 11A is highly prevalent in Korea. We investigated the prevalence and characteristics of Streptococcus pneumoniae serotype 11A isolates in a Korean tertiary-care hospital, during 2004–2013. A total of 1579 non-duplicate clinical S. pneumoniae isolates, collected from 2004 to 2013, were included in this study. Serotype was determined by the capsular Quellung method, and in vitro susceptibility testing was performed by broth microdilution method. Multilocus sequence typing was performed to determine the genotypes of the S. pneumoniae isolates. We identified 90 serotype 11A isolates (5.7%). During this period, the proportion of serotype 11A has increased from 3.2% up to 13.2% (in 2012). Among the serotype 11A isolates, two main clonal complexes (CCs), CC166 and CC99, were identified. The increase of serotype 11A was mainly due to the increase of CC166 isolates, which have high antimicrobial resistance rates. In addition, we identified that 14 isolates, belonging to ST8279, ST9875, and ST3598 of CC166, were non-susceptible to all antimicrobial agents tested in this study. We identified the increase of S. pneumoniae serotype 11A in Korea, which mainly due to the expansion of a resistant clonal group, CC166.

Longitudinal study on Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus nasopharyngeal colonization in HIV-infected and -uninfected infants vaccinated with pneumococcal conjugate vaccine

BackgroundStreptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus are all potentially pathogenic, which frequently colonize the nasopharynx (NP) prior to causing disease.We studied bacterial NP-colonization in 321 HIV-infected and 243 HIV-uninfected children vaccinated with 7-valent pneumococcal conjugate vaccine (PCV7) at 6, 10 and 14 weeks of age. MethodsHIV-uninfected infants included those born to HIV-uninfected (HUU) and HIV-infected women (HEU); HIV-infected children with CD4+ lymphocyte ≥25% were randomized to initiate antiretroviral therapy immediately (ART-Immed) or when clinically indicated (ART-Def). Nasopharyngeal swabs for bacterial culture were taken prior to each PCV7 dose (Visits 1–3) and at 20, 39, 47 and 67 weeks of age (Visits 4–7). Swabs were cultured by standard methods and pneumococcal serotyping done by the Quellung method. ResultsColonization patterns for pneumococcus, H. influenzae and S. aureus did not differ between HUU and HEU children; and were also generally similar between ART-Def and ART-Immed children. Prevalence of PCV7-serotype colonization was similar between HIV-infected and HIV-uninfected children, however, overall pneumococcal and specifically non-vaccine serotype colonization tended to be lower in HIV-infected children. HIV-infected children also had a 44% lower prevalence of S. aureus colonization at Visit-1 (p=0.010); and H. influenzae colonization was also lower among HIV-infected than HIV-uninfected children at Visit-2, Visit-3, Visit-6 and Visit-7. ConclusionVaccine-serotype colonization is similar in PCV-immunized HIV-infected and HIV-uninfected children. We, however, identified a lower prevalence of overall-pneumococcal and H. influenzae colonization in HIV-infected children post-PCV vaccination, the clinical-relevance of which warrants further study.

Acquisition of Streptococcus pneumoniae in South African children vaccinated with 7-valent pneumococcal conjugate vaccine at 6, 14 and 40 weeks of age

BackgroundSeven-valent pneumococcal conjugate vaccine (PCV7) was introduced into the South African immunization program using 6, 14 and 40 weeks dosing schedule (2+1), with no catch-up in older children since April 2009. We investigated pneumococcal colonization acquisition in children who received this schedule and also compared it to historical cohorts of PCV-naïve children (n=123 in 2007) and children who received a 3+1 PCV7 schedule (n=124 in 2005/06). MethodsTwo hundred and fifty children aged 6–12 weeks were enrolled from December 2009 to April 2010. Participants had nasopharyngeal swabs collected on eight occasions between enrolment and 2-years of age. Standard methods were undertaken for bacterial culture and Streptococcus pneumoniae were serotyped using the Quellung method. Pneumococcal and Staphylococcus aureus colonization in the present study was compared to colonization in two historical longitudinal cohorts. ResultsS. pneumoniae was identified in 1081 (61.4%) of 1761 swabs collected in the current cohort. Pneumococcal colonization peaked at 41-weeks of age (76.8%) and decreased to 62.8% by 2-years of age (p=0.002); PCV7-serotype colonization decreased during the same period from 28.6% to 15.6% (p=0.001). Children from the current cohort compared to PCV-naïve children were less likely to be colonized by PCV7-serotypes from 40-weeks to 2-years of age and acquired PCV7-serotypes less frequently. No differences in overall pneumococcal, PCV7-serotype and non-PCV7-serotype colonization or new serotype acquisitions were detected comparing the current cohort to the historical cohort who received the 3+1 PCV7 schedule. Staphylococcus aureus colonization was similar in all three cohorts. ConclusionA 2+1 PCV7 schedule implemented in South Africa was temporally associated with reduced risk of vaccine-serotype colonization compared to historically unvaccinated children. Also, vaccine-serotype acquisition rate using the 2+1 schedule was similar to that in the 3+1 dosing cohort, suggesting that similar indirect protection against pneumococcal disease could be derived from either schedule in South Africa.

Serotyping of Streptococcus pneumoniae Based on Capsular Genes Polymorphisms

Streptococcus pneumoniae serotype epidemiology is essential since serotype replacement is a concern when introducing new polysaccharide-conjugate vaccines. A novel PCR-based automated microarray assay was developed to assist in the tracking of the serotypes. Autolysin, pneumolysin and eight genes located in the capsular operon were amplified using multiplex PCR. This step was followed by a tagged fluorescent primer extension step targeting serotype-specific polymorphisms. The tagged primers were then hybridized to a microarray. Results were exported to an expert system to identify capsular serotypes. The assay was validated on 166 cultured S. pneumoniae samples from 63 different serotypes as determined by the Quellung method. We show that typing only 12 polymorphisms located in the capsular operon allows the identification at the serotype level of 22 serotypes and the assignation of 24 other serotypes to a subgroup of serotypes. Overall, 126 samples (75.9%) were correctly serotyped, 14 were assigned to a member of the same serogroup, 8 rare serotypes were erroneously serotyped, and 18 gave negative serotyping results. Most of the discrepancies involved rare serotypes or serotypes that are difficult to discriminate using a DNA-based approach, for example 6A and 6B. The assay was also tested on clinical specimens including 43 cerebrospinal fluid samples from patients with meningitis and 59 nasopharyngeal aspirates from bacterial pneumonia patients. Overall, 89% of specimens positive for pneumolysin were serotyped, demonstrating that this method does not require culture to serotype clinical specimens. The assay showed no cross-reactivity for 24 relevant bacterial species found in these types of samples. The limit of detection for serotyping and S. pneumoniae detection was 100 genome equivalent per reaction. This automated assay is amenable to clinical testing and does not require any culturing of the samples. The assay will be useful for the evaluation of serotype prevalence changes after new conjugate vaccines introduction.

Microbiological characterization of Streptococcus pneumoniae and non-typeable Haemophilus influenzae isolates as primary causes of acute otitis media in Bulgarian children before the introduction of conjugate vaccines

BackgroundPneumococcal and Haemophilus influenzae type b (Hib) vaccines were introduced in our national immunisation program in April 2010. The aims of this retrospective, laboratory-based study were to determine the serotypes and antibiotic resistance of Streptococcus pneumoniae and H. influenzae isolates from middle ear fluid (MEF) collected before the introduction of immunization.MethodsS. pneumoniae (n = 128) and H. influenzae (n = 40) strains isolated from MEF of children with AOM between 1994 and 2011 were studied. MICs were determined by a microdilution assay. Serotyping of S. pneumoniae was done by Quellung method and PCR capsular typing was used for H. influenzae. Macrolide resistance genes were detected by PCR for erythromycin resistant S. pneumoniae (ERSP). DNA sequencing of ftsI gene was performed for ampicillin nonsusceptible H. influenzae.ResultsThe most common serotypes found among children with pneumococcal AOM were 19 F (20.3%), 6B (15.6%), and 19A (10.9%). The potential coverage rates by the PCV7, PCV10 and PCV13 of children aged < 5 years were 63.6%, 66.4% and 85.5%, respectively. Reduced susceptibility to oral penicillin was seen in 68.1%; resistance to erythromycin was 46.9%. We found erm(B) gene in 56.7% of the ERSP, mef(E) gene in 25%; 15% harbored both genes erm(B) + mef(E) and 3.3% had mutations of L4 ribosomal protein. Of the 40 H. influenzae isolates 97.5% were nontypeable. Nonsusceptibility to ampicillin occurred in 25%. Ampicillin resistance groups were: β-lactamase-positive ampicillin resistant (BLPAR) strains (10%), β-lactamase-negative ampicillin resistant (BLNAR) strains (12.5%) and β-lactamase-positive amoxicillin-clavulanate resistant (BLPACR) strains (2.5%). Among BLNAR and BLPACR most of the isolates (5/6) belonged to group II, defined by the Asn526Lys substitution.ConclusionsThe levels of antibiotic resistance among S. pneumoniae and H. influenzae causing severe AOM in children are high in our settings. The existence of multidrug-resistant S. pneumoniae serotype 19A is of particular concern. The rate of BLNAR and BLPACR strains among H. influenzae isolates was 15%.

Baseline Epidemiology of Streptococcus pneumoniae Serotypes in Canada Prior to the Introduction of the 13-Valent Pneumococcal Vaccine

Changes in the epidemiology of Streptococcus pneumoniae were reported worldwide after the introduction of the 7-valent pneumococcal vaccine, particularly an increase in multi-drug resistant (MDR) 19A strains. Subsequently, a 13-valent pneumococcal vaccine (PCV-13) has been introduced. This study assessed the incidence of S. pneumoniae serotypes in all age groups prior to the introduction of PCV-13 in Canada (2007-2009). Eight hundred S. pneumoniae isolates from respiratory specimens and blood cultures were collected as part of a Canadian surveillance study (CANWARD) from patients in 15 tertiary-care centers. Serotyping was performed by the Quellung method and antimicrobial susceptibility testing was performed by broth microdilution in accordance with the Clinical and Laboratory Standards Institute guidelines. The most common serotypes were 19A (8.6%), 3 (7.3%), 22F (6.0%), 4 (4.6%), 5 (4.4%), and 11A (4.4%); and the first serotype 6D isolate in Canada was identified. Serotypes 5, 7F, and 19A were significantly (p<0.001) more frequently isolated from bloodstream infections. Considerable serotype variability was noted for different age groups: 15B (p<0.01) and 19A (p<0.001) were more frequently isolated from children ≤2 years old. Overall, 46.4% of currently circulating S. pneumoniae serotypes in Canada are included in PCV-13. Notably, 87.5% of MDR-S. pneumoniae were covered by PCV-13. Accordingly, PCV-13 will provide coverage against a significant proportion of circulating S. pneumoniae strains in Canada, including the critical antimicrobial-resistant strains.

Comparison of methods for detection of Streptococcus pneumoniae serotype 6C

Polymerase chain reaction-based methods and Quellung method were compared for serotyping of Streptococcus pneumoniae serogroup 6 for 230 isolates from Asian countries. Prevalence of serotype 6C differed according to the method used, with inconsistencies in 12 pneumococcal isolates (5.2%). Thus, evaluation of the prevalence of serotype 6C may differ according to the methods used.

Multiplex PCR reveals a high rate of nasopharyngeal pneumococcal 7-valent conjugate vaccine serotypes co-colonizing indigenous Warao children in Venezuela

Knowledge of co-colonization with multiple pneumococcal serotypes is becoming very important in the light of both serotype replacement and switching as a result of vaccination. Co-colonization has been reported to occur in up to 30% of carriers, especially in populations with high Streptococcus pneumoniae carriage rates. For the determination of co-colonization, single colonies of nasopharyngeal specimens are serotyped with the Quellung method, a costly method with a low sensitivity. Here we explore the use of a multiplex PCR to identify simultaneous carriage of the capsular serotypes targeted by the 7-valent conjugate vaccine. We applied this multiplex PCR to 50 primary cultures from the nasopharyngeal swabs of healthy Warao Amerindian children, a population with a high pneumococcal carriage rate, most of them with vaccine serotypes, and we identified a second serotype in 20% (n=10) of the pneumococci carriers. These results were confirmed by detailed serotyping of multiple colonies isolated from the primary culture with the Quellung method. We conclude that the multiplex PCR is a sensitive, simple and cost-effective method for detecting multiple serotypes in nasopharyngeal cultures, and thus might be useful for the monitoring of pneumococcal colonization over time, especially in the surveillance of nasopharyngeal colonization after conjugate vaccination.

Changes of serotype and genotype in Streptococcus pneumoniae isolates from a Korean hospital in 2007

We investigated the change in clones and serotypes of Streptococcus pneumoniae isolates in a Korean tertiary-care hospital. Serotypes of S. pneumoniae isolates were determined by the capsular quellung method, and in vitro susceptibility testing was performed by broth microdilution method. Multilocus sequence typing was performed to determine the genotypes of the S. pneumoniae isolates. The erm(B) and mef(A) genes in erythromycin-resistant isolates were also detected using the duplex polymerase chain reaction method. During the 2 periods assayed (1998–2000 and 2007), 7-valent pneumococcal conjugate vaccine (PCV7) serotypes decreased significantly from 58.3% to 30.9% ( P = 0.001). Especially, serotypes 19F and 23F decreased significantly from 31.7% to 8.5% ( P < 0.0001) and from 20.0% to 7.4% ( P = 0.021), respectively. In contrast to the other PCV7 serotypes, serotype 14 coupled with CC554 emerged in 2007, which may indicate no effect of PCV7 against serotype 14 isolates from Korea and the possibility of a different subtype. Of the non- PCV7 serotypes, serotype 19A increased from 8.3% to 14.9% ( P = 0.227) and serotype 15 increased from 0% to 8.5% ( P = 0.023). The increase of serotype 19A was due to the expansion of a preexisting clone with serotype 19A, ST320. However, S. pneumoniae isolates of serotype 15 showed diverse STs. Our data may provide helpful information in local vaccine serotype expansion or replacement in Korea.

FIELD EVALUATION OF THE CHESSBOARD MODIFICATION FOR SEROTYPING OF STREPTOCOCCUS PNEUMONIAE IN A SMALL LABORATORY IN BANGLADESH

The chessboard modification of the quellung method for serotyping Streptococcus pneumoniae (Pneumococcus) was introduced in the Gonoshasthaya Vaccine Research Laboratory, a small laboratory in Bangladesh. We applied initial bench-side training and subsequent continuous surveillance and quality assurance as approaches for good laboratory practice. Results obtained a this laboratory on 1,101 consecutive isolates had satisfactory sensitivity (85.1-100%) and specificity (97.9-99.9%) for serotyping the 10 most common serogroups/types of pneumococci when compared with the results obtained in the Finnish reference laboratory for pneumococcal serotyping at the National Public Health Institute. We conclude that serotyping of pneumococci by the chessboard method can be introduced into a small laboratory by providing basic bacteriologic skills, adequate initial training, and continuous external support.