Enhancing sensitivity of qPCR assays targeting Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae by using a mutant Taq DNA polymerase.

AIMS: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae are important causes of bacterial meningitis. In this study, the DNA binding site of the wild type Taq DNA polymerase was modified to produce a mutant enzyme with enhanced DNA affinity and PCR performance. The engineered and the wild type enzymes were integrated into qPCR-based assays for molecular detection of S. pneumoniae, N. meningitidis, H. influenzae, and serogroups and serotypes of these three pathogens. METHODS: Bio-Speedy® Bacterial DNA Isolation Kit (Bioeksen R&D Technologies, Turkiye) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Turkiye) and CFX96 Instrument (Biorad Inc., USA) were used for all molecular analyses. Spiked negative clinical specimens were tested using the developed qPCR assays and the culture-based conventional methods for the analytical performance evaluation. RESULTS: All qPCR assays did not produce any positive results for the samples spiked with potential cross-reacting bacteria. Limit of detection (LOD) of the assays containing the mutant enzyme was 1 genome/reaction (10 cfu/mL sample) which is at least 3 times lower than the previously reported LOD levels for DNA amplification based molecular assays. LODs for the spiked serum and cerebrospinal fluid (CSF) samples decreased 2.3-4.7 and 1.2-3.5 times respectively when the mutant enzyme was used instead of the wild type Taq DNA polymerase. CONCLUSIONS: It is possible to enhance analytical sensitivity of qPCR assays targeting the bacterial agents of meningitis by using an engineered Taq DNA polymerase. These qPCR-based assays can be used for direct detection and serogrouping / serotyping of S. pneumoniae, N. meningitidis and H. influenzae at concentrations close to the lower limit of medical decision point.

[Legionella Species Isolated in the Water Systems of Ships and Their Molecular Characterization]. / Gemilerin Su Sistemlerinden Izole Edilen Legionella Türleri ve Moleküler Karakterizasyonu.

Legionella species are generally found in nature and in water resources, and they are gram negative bacilli that can cause pneumonia by being transmitted from water systems to humans via aerosol or aspiration. Legionnaires' disease caused by this agent continues to be a public health problem in cruise ships. In this study, it was aimed to determine the prevalence of the colonization of Legionella species by culture method and to determine the molecular characterization of the isolated Legionella in water samples taken from the water systems of the ships docking in Mersin International Port. A total of 158 cold water samples were taken from 18 ferry and/or cargo ships docking in Mersin International Port between December 2014 and June 2015. Fifty-four of the samples were obtained from tanks, 68 from taps and 36 from shower heads. All samples were centrifuged and inoculated from the pellet onto "Buffered Coal Yeast Extract" (BCYE) (Oxoid, CM0655, UK) agar medium supplemented with iron pyrophosphate, L-cysteine and α-ketoglutarate (Oxoid, SR0110, UK). The culture plates were incubated for 10-15 days in microaerophilic environment in a desiccator at 37°C. The suspicious colonies grown in cultures were serogrouped by latex agglutination test (Oxoid, DR0800M, UK) and fluorescent antibody method (m-Tech Monoclonal Technologies, Inc., USA). For the molecular analysis of Legionella species grown in culture, DNA isolation was made from Legionella colonies and then polymerase chain reaction amplification was performed using specific primer sequences targeting the rpoB gene region of the Legionella genome. Direct DNA sequencing of rpoB gene products was performed in the "ABI PRISM 3130XL Genetic Analyzer" (Applied Biosystems, USA). The DNA sequences were typed by BLAST analysis and the determined types, and NCBI (National Center for Biotechnology Information) reference Legionella sequences were phylogenetically compared with the Neighbor-Joining comparison method by using the Mega 7 program. Legionella spp. was isolated in 18 (11.4%) of 158 samples. Of these, four (7.4%, 4/54) were detected from the tank, 11 (16.2%, 11/68) from the tap and three (8.33%, 3/36) from the shower head. After the latex agglutination test performed from the growing bacterial colonies, five (27.8%) were serogrouped as Legionella spp., four (22.2%) as Legionella pneumophila sg 5, two (11.1%, each) as L.pneumophila sg 1,L.pneumophila sg 8 and Legionella bozemanii and one (5.6%) as L.pneumophila sg 3. Two (11.1%) of the isolates grown in culture could not be serogrouped. Molecular characterization of 12 Legionella isolates could be performed. One of them was serologically serogrouped as L.bozemanii, and it was found to be 99% similar to Legionella rubrilucens when compared with NCBI Legionella sequence data in the BLAST program. One isolate that could not be differentiated by serogrouping was identified as Legionella erytra in the BLAST program after DNA sequence analysis. The remaining 10 isolates (55.6%, n= 18) were confirmed as L.pneumophilia after the comparison with reference NCBI sequences. In this study, it was determined that 11.4% of the water samples collected from the water systems of the ships docking in Mersin International Port were contaminated with Legionella species. The detected Legionella species have an important potential source of infection for the captain, ship workers and passengers travelling on the ships. In this respect, this study reveals the necessity of establishing studies to improve the risk management of Legionella in the water systems of ships.

[Burden of Pneumococcal Meningitis and Bacteremia, Serotype Distribution and Antibiotic Resistance in Healthy Children After Conjugated Pneumococcal Vaccine Implementation: Single Center Experience]. / Saglikli Çocuklarda Konjuge Pnömokok Asisi Uygulamasi Sonrasi Pnömokokal Menenjit ve Bakteriyemi Hastalik Yükü, Serotip Dagilimi ve Antibiyotik Direnci: Tek Merkez Deneyimi.

In Turkey, the seven-valent pneumococcal conjugated vaccine (PCV7) was included in the childhood national immunization programme in April 2008 and was replaced by the 13-valent pneumococcal conjugated vaccine (PCV13) in April 2011. In this retrospective, single-center study, it was aimed to determine the serotype distribution and antimicrobial resistance in Streptococcus pneumoniae isolates of pediatric patients with invasive pneumococcal disease (IPD) after the introduction of PVC7 and PVC13. Fifty pediatric patients diagnosed with meningitis and sepsis/bacteremia between October 2009 and October 2019 were included in the study. The pediatric patient group consisted of previously healthy patients diagnosed with meningitis and sepsis/bacteremia with S.pneumoniae isolated in their blood or cerebrospinal fluids. Patients with pneumonia-associated bacteremia and empyema were not included in the study. Serotyping of the isolates was performed by Quellung reaction using specific antisera (Statens Serum Institute, Denmark) and antibiotic (penicillin and ceftriaxone) susceptibility was determined by antibiotic gradient method based on Clinical Laboratory Standards Institute (CLSI) criteria. Of the children, 29 (58%) were boys and 21 (42%) were girls. The median age of the patients was 19 months (1 month-18 year). When the children under the age of five were evaluated, it was found that 30 (79%) patients were diagnosed with occult bacteremia/sepsis and 8 (21%) with meningitis. The overall annual incidence rate of IPD among the healthy children aged <5 years decreased significantly from 9.35/100000 to 0.83/100000 (p< 0.001). Serotype identification was determined for 44 of 50 pneumococcal isolates . However, since six patients with underlying disease were not included in the evaluation, the remaining 38 isolates were found to be one of the serotypes included in PCV7 and PCV13 at a rate of 28.9% (n= 11) and 44.7% (n= 17), respectively. While the rate of PCV13 serotypes seen in the PCV7 period was 81.8%, this rate decreased to 29.6% within eight years after PCV13 administration. The rate of non-vaccine serotypes was determined as 54.5% in PCV7 period and 70.3% in PCV13 period. The rate of non-vaccine serotypes in patients under 5 years was 60% in the period of PCV7 and 75% in the period of PCV13. The proportion of non-vaccine serotypes has increased over time. However, this difference was not statistically significant (p> 0.05). The most common serotypes detected in isolates were 19F, 23F, 7F, 31 and 24B. According to the minimum inhibitory concentration values of the isolates recovered from patients with meningitis, penicillin and ceftriaxone resistance rates were found as 43.9% and 9.8%, respectively. In conclusion, our study showed that there was a 91.1% decrease in the incidence of IPD in healthy children aged under five years after the implementation of PCV7 and PCV13. It was determined that while the rate of serotypes in vaccine content decreased, there was an increase in non-vaccine serotypes. In addition no significant change was observed in antibiotic resistance rates over the years.

Vaccine failures in pediatric cases caused by streptococcus pneumoniae serotype 19A.

Community-acquired pneumonia (CAP) is a leading cause of childhood mortality and morbidity worldwide. PCV-13 has implemented incidence of CAP undoubtedly in a very good way but continuous surveillance for vaccine failures is still very important for future vaccination programs. To support this opinion we report seven children (age of the seven patients ranged between 10 months and 5 y, 10 months old patients were vaccinated 3 times with PCV13 whereas others were fully vaccinated as 3 + 1) infected with Streptococcus pneumoniae Serotype 19A in last 4 y (2015-2018).

A Syrian refugee with meningococcal empyema.

Meningococcal infections are an important cause of mortality and morbidity in childhood. Herein we report a 2-year-old Syrian refugee boy living in Turkey diagnosed with Neisseria meningitidis serotype B infection complicated with intracranial empyema. Refugees are a sensitive population in all aspects especially infectious diseases. Endemic diseases in the refugees' own countries constitute a major health problem. Vaccination in sensitive populations and in the country would be important precaution to decrease disease burden.

The impact of meningococcal conjugate vaccine (MenACWY-TT) on meningococcal carriage in Hajj Pilgrims returning to Turkey.

Meningococcal conjugate vaccine was administered for the first time to pilgrims in 2018 in Turkey prior to their departure to the Hajj. This study aimed to determine the effect of vaccination on the prevalence of Neisseria meningitidis carriage among the pilgrims returning to Turkey. This prospective paired (departing and arriving) cohort study included pilgrims aged 10-80 years and compared N.meningitidis carriage before and after pilgrimage. Oropharyngeal samples were collected from 229 pilgrims before departing for Mecca, Kingdom of Saudi Arabia (KSA) and after their return to Turkey. Meningococcal carriage was detected in 3.9% (n = 9) at the time of departure; all positive samples were serogroup B. Upon returning to Turkey,1 (0.4%) pilgrim, who was not a carrier before pilgrimage, was positive for serogroup B. This study is the first to examine meningococcal carriage following the administration of conjugate vaccine to Hajj pilgrims from Turkey.

Pediatric cases caused by Streptococcus pneumoniae Serotype 3.

Streptococcus pneumoniae causes invasive pneumococcal disease (IPD), leading to high morbidity and mortality worldwide. Although nonvaccine serotypes constitute a major issue in the aspect of invasive pneumococcal disease, serotypes included in PCV13 still continue to be a problem. Vaccination with very high coverage and multicenter surveillance studies would be beneficial to decrease carriage of vaccine and nonvaccine serotypes of pneumococcus and monitor changing seroepidemiology.

Impact of PCV7 vaccination on nasopharyngeal carriage and antimicrobial resistance among children in Turkey.

INTRODUCTION: We aimed to evaluate the effects of 7-valent pneumococcal conjugate vaccine on nasopharyngeal carriage of Streptococcus pneumoniae and antibiotic resistance in children in a well-child clinic in a tertiary children's hospital in Turkey. METHODOLOGY: We collected nasopharyngeal (NP) specimens from 557 two-month-old babies before vaccination. After the study population had received PCV7, NP samples were obtained from 135 babies. Antimicrobial susceptibility testing and serotyping were performed. RESULTS: S. pneumoniae colonized in 48 (8.6%) of the 557 two-month-old babies before vaccination. The follow-up cohort consisted of 135 subjects. The prevalence of PCV7 strain decreased from 33.3% to 19.3% after vaccination. However, non-PCV7 types increased from 66.6% to 80.6% (p = 0.02). Of PCV7 serotypes, 19F was the most frequent serotype before and after vaccination. There was an increase in 6A and 15 of non-PCV7 serotypes after vaccination. Penicillin non-susceptible increased from 56.3% to 80.6% after vaccination (p =0.03). Serotypes 14, 18C, 9V and 6B, which were identified before vaccination, never colonized afterwards. Number of siblings and having sibling with older age of five were determined to be significant effective factors for SP colonization presence after vaccination and antibiotic use was negatively associated with pneumococcal carriage but associated with penicillin non-susceptibility. CONCLUSIONS: Nasopharyngeal carriage rate of S. pneumoniae dropped after PCV7 vaccination, and replacement by NVT pneumococci were also observed. Risk factors for nasopharyngeal carriage included household crowding and having a sibling age five years or older. Penicillin non-susceptibility increased in both VT and NVT strains.

[Optimization of real-time multiplex polymerase chain reaction for the diagnosis of acute bacterial meningitis and Neisseria meningitidis serogrouping]. / Akut bakteriyel menenjit tanisi ve Neisseria meningitidis serogruplandirmasinda gerçek zamanli multipleks polimeraz zincir reaksiyonunun optimizasyonu.

Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae type B are responsible for about 80-85% of acute bacterial meningitis cases among adults and in our country it is mandatory to report bacterial meningitis cases caused by these agents. In recent years, disease control programs have focused especially on these three pathogens in our country. It is very important to know the causative agent of meningitis for prevention/control measures, choice of treatment regimen, and prediction of the severity and the course of the disease. Rapid diagnosis of meningococcal meningitis is especially vital. In addition, the identification of N.meningitidis serogroups in the community is also important for the decision of the type and combination of vaccine to be administered. The aim of this study was to optimize the real-time multiplex polymerase chain reaction (Rt-multiplex PCR) for the diagnosis of the disease and serogrouping of N.meningitidis in clinical samples in a direct, quick and reliable way. In this study, three Rt-multiplex PCR assays were optimized and standardized for the detection of N.meningitidis, H.influenzae and S.pneumoniae (NHS mix) and for the serogrouping of N.meningitidis (BCY mix and AWX mix) in our laboratory. Sensitivity of the Rt-multiplex PCR method was detected by reference strains and simulation studies. Forty three samples (41 cerebrospinal fluid (CSF), 1 serum and 1 clinical isolate) with the suspicion of acute bacterial meningitis and six nasopharyngeal isolates sent to National Reference Laboratory for Molecular Microbiology between July 2012-May 2014 were included in the present study. All samples were examined with the Rt-multiplex PCR methods. Clinical isolates were studied by both conventional and Rt-multiplex PCR methods. Oxidase, catalase test, Gram stain, API-NH and agglutination tests with specific antisera were performed in the National Respiratory Pathogens Reference Laboratory. The detection limit of the method, which was optimized and standardized in our laboratory was determined as 102 cfu/ml. The CT (threshold cycle) value in this dilution was detected approximately as 35. N.meningitidis was detected in 14 of the 41 CSF samples by the NHS mix. However, only 10 of the positive samples could be typed with BCY mix and AWX mix. Eight (80%) of them were serogroup B, one of each was (10%) serogroup A and serogroup W135, respectively. All the isolates (six nasopharyngeal and one clinical specimen) were identified as N.meningitidis serogroup B by Rt-multiplex PCR and the isolates were also confirmed by conventional methods. The CSF specimens with CT value > 35 could not be typed. We concluded that the Rt-multiplex PCR method is a rapid and reliable test for the direct diagnosis of acute bacterial meningitis due to NHS and serogrouping of N.meningitidis. Rapid diagnosis plays an important role for the treatment and control of the disease, and serogrouping of the agent plays an important role in terms of prevention/control and vaccination policies.

[Screening of Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis in throat swab specimens of children with upper respiratory tract infections]. / Üst solunum yolu enfeksiyonu bulgulari ile basvuran çocuklarin bogaz sürüntü örneklerinde Corynebacterium diphtheriae, Corynebacterium ulcerans ve Corynebacterium pseudotuberculosis suslarinin arastirilmasi.

Although a significant decrease has been reported in the incidence of diphteria in many regions of the world following the routine diphtheria immunization programs, the emergence of new cases indicated that toxigenic strains are still circulating in the community. Diphtheria vaccine does not provide protection against asymptomatic carriage and colonization of non-toxigenic Corynebacterium diphtheriae. It is a known fact that invasive infections may arise from non-toxigenic C.diphtheriae strains that the non-toxigenic strains can become toxigenic strains leading to diphteria. It is also known that there is a risk of diphteria outbreaks due to decreased antitoxin level and inadequate adult immunization programs. In our country, there is no routine surveillance of toxigenic and non-toxigenic C.diphtheriae. In the present study we aimed to investigate the presence of C.diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis in children presenting with the symptoms of upper respiratory tract infections that might be confused with those moderate diphteria, in order to highlight the requirement of microbiological surveillance and to create awareness about these microorganisms among public health experts, microbiologists and clinicians. Throat swab specimens were obtained from children who were admitted to the pediatric outpatient clinics, in Dr. Sami Ulus Obstectrics, Children Health and Diseases Educational and Research Hospital, with upper respiratory tract infections between 1 February 2016-22 March 2016. The specimens were inoculated in 5% sheep blood agar plates. The plates that were incubated in appropriate conditions, were evaluated for Group A beta hemolytic streptococcocci. Subsequently, culture plates were sent to the Public Health Institution of Turkey, National Respiratory Pathologens Reference Laboratories for the investigation of the presence of C.diphtheriae, C.ulcerans and C.pseudotuberculosis. The growth in each plate were collected with a sterile swab and inoculated in tryptic soy broth. Following 2 hours of incubation at 37oC, subcultures were inoculated in cystine-tellurite-blood agar (CTBA) and 5% sheep-blood agar plates; after an overnight incubation tellurite-reducing colonies were inoculated in Tinsdale agar plates. The suspected colonies with positive cystinase activity were identified by conventional methods and also with Coryne API (Biomerieux, France) systems. Toxicity tests (ELEK, PCR) were performed to investigate whether the C.diphtheriae strains were producing toxins. A total of 500 patients were involved in the study. Of these 260 (52%) were girls and 240 (48%) were boys with a mean age of 76 (range, 21-213) months. All patients except one were fully vaccinated with boosters. Most common presenting symptoms of the patients were fever (19.8%), sore throat (52.6%), cough (49.2%), tonsillar hyperemia (97.6%), presence of crypt (24.6%), and membrane over tonsils (1%). Group A beta-hemolytic streptococcocci were detected in the throat swab cultures of 66 (%13.2) patients. Genotypically toxin negative C.diphtheriae biovar gravis was identified in the throat swab cultures of 3 patients (2 girls and 1 boy). The tonsils were hyperemic and hypertrophic in all the patients with C.diphtheriae biovar gravis. C.ulcerans and C.pseudotuberculosis were detected in none of the patients. It is considered that similar regular cross-sectional studies or routine screening programs are expected to raise awareness about this forgotten microorganism both epidemiologically and microbiologically.

Efectos de la vacuna neumocócica conjugada (VCN7 y VCN13) en los niños turcos con enfermedad neumocócica invasiva: experiencia en un solo centro / The effects of pneumococcal conjugate vaccine (PCV7 and PCV13) on Turkish children with invasive pneumococcal disease: a single center experience

Introducción: Nuestro objetivo fue determinar los cambios en la incidencia de enfermedad neumocócica invasiva (ENI), la distribución de serotipos y patrones de resistencia antibiótica del Streptococcus pneumoniae en niños con ENI tras el período de vacunación (de 1 a 7 años) con vacuna neumocócica de 7 serotipos (VCN7) (2008) y de 13 serotipos (VCN13) (2011). Población y métodos: El estudio se realizó en 39 niños con ENI de 1 mes a 18 años de edad en Angora, Turquía. Se identificó Streptococcus pneumoniae en sangre, líquido cefalorraquídeo, líquido pleural, y otros tejidos y líquidos corporales estériles mediante procedimientos estándar. Se analizó la resistencia de cepas aisladas de S. pneumoniae a penicilina y ceftriaxona con la prueba de epsilometría (E-test). Los serotipos de las cepas se determinaron con la reacción de Quellung. Resultados: La incidencia anual de ENI disminuyó significativamente de 7,71 (intervalo de confianza --#91;IC--#93; del 95%: de 1,99 a 13,4) a 1,58 (IC del 95%: de 0,6 a 3,77; reducción del riesgo relativo= -79,5; p= 0,006) cada 100 000 habitantes de < 5 años de edad sin enfermedad preexistente. Durante todo el período del estudio, los serotipos en la VCN7 y en la VCN13 representaron el 27,8% y el 63,8% de las cepas aisladas, respectivamente. Los serotipos en la VCN13 correspondían al 81,8% de los casos de ENI en la era previa a la introducción de esta vacuna, y disminuyeron al 56% en los cuatro años posteriores. Las tasas de resistencia a penicilina y ceftriaxona (en el caso de la meningitis) fueron del 48,5% y el 9,1%, respectivamente. Conclusiones: Este estudio observó una disminución significativa en la incidencia de ENI después de la introducción de la VCN13.

Introduction. The aim of this prospective singlecenter study was to determine the changings in incidence of invasive pneumococcal disease (IPD), serotype distribution and the antimicrobial resistance patterns of S. pneumoniae in children with IPD after the period (1 to 7 years) of vaccination with PCV7 (2008) and PCV13 (2011). Population and methods. The study was conducted on 39 Turkish children with IPD between ages 1 month and 18 years in Ankara, Turkey. Streptococcus pneumoniae was identified using standard laboratory procedures from blood, cerebrospinal fluid (CSF), pleural fluid, and other sterile body fluids and tissues. S. pneumoniae isolates were tested for resistance to penicilin and ceftriaxone using the E-test methodology. Serotypes of the isolates were determined by Quellung reaction. Results. The overall annual incidence rate of IPD decreased significantly from 7.71 (95% CI, 1.99-13.4) to 1.58 (95% CI, 0.6-3.77; RRR= -79.5; p= 0.006) per 100 000 population among <5 years of age without underlying disease. During the overall study period, the PCV7-serotypes and PCV13-serotypes represented 27.8% and 63.8% of isolates, respectively. PCV13-serotypes made up 81.8% of cases of IPD in the pre-PCV13 era and decreased to 56% in the 4 years after PCV13. The penicillin and ceftriaxone (for meningitis) resistance rates were 48.5% and 9.1%, respectively. Conclusions. This is the first study about the changing pattern of the incidence of IPD in Turkish children after the implementation of the PCV7 and PCV13 in Turkish national vaccine schedule and a prominent decrease in incidence of IPD has seen after the implementation of PCV13.

The effects of pneumococcal conjugate vaccine (PCV7 and PCV13) on Turkish children with invasive pneumococcal disease: a single center experience. / Efectos de la vacuna neumocócica conjugada (VCN7 y VCN13) en los niños turcos con enfermedad neumocócica invasiva: experiencia en un solo centro.

INTRODUCTION: The aim of this prospective single-center study was to determine the changings in incidence of invasive pneumococcal disease (IPD), serotype distribution and the antimicrobial resistance patterns of S. pneumoniae in children with IPD after the period (1 to 7 years) of vaccination with PCV7 (2008) and PCV13 (2011). POPULATION AND METHODS: The study was conducted on 39 Turkish children with IPD between ages 1 month and 18 years in Ankara, Turkey. Streptococcus pneumoniae was identified using standard laboratory procedures from blood, cerebrospinal fluid (CSF), pleural fluid, and other sterile body fluids and tissues. S. pneumoniae isolates were tested for resistance to penicilin and ceftriaxone using the E-test methodology. Serotypes of the isolates were determined by Quellung reaction. RESULTS: The overall annual incidence rate of IPD decreased significantly from 7.71 (95% CI, 1.99-13.4) to 1.58 (95% CI, 0.6-3.77; RRR=-79.5; p=0.006) per 100 000 population among ≤5 years of age without underlying disease. During the overall study period, the PCV7-serotypes and PCV13-serotypes represented 27.8% and 63.8% of isolates, respectively. PCV13-serotypes made up 81.8% of cases of IPD in the pre-PCV13 era and decreased to 56% in the 4 years after PCV13. The penicillin and ceftriaxone (for meningitis) resistance rates were 48.5% and 9.1%, respectively. CONCLUSIONS: This is the first study about the changing pattern of the incidence of IPD in Turkish children after the implementation of the PCV7 and PCV13 in Turkish national vaccine schedule and a prominent decrease in incidence of IPD has seen after the implementation of PCV13.

INTRODUCCIÓN: Nuestro objetivo fue determinar los cambios en la incidencia de enfermedad neumocócica invasiva (ENI), la distribución de serotipos y patrones de resistencia antibiótica del Streptococcus pneumoniae en niños con ENI tras el período de vacunación (de1 a 7 años) con vacuna neumocócica de 7 serotipos (VCN7) (2008) y de 13 serotipos (VCN13) (2011). POBLACIÓN Y MÉTODOS: El estudio se realizó en 39 niños con ENI de 1 mes a 18 años de edad en Angora, Turquía. Se identificó Streptococcus pneumoniae en sangre, líquido cefalorraquídeo, líquido pleural, y otros tejidos y líquidos corporales estériles mediante procedimientos estándar.Se analizó la resistencia de cepas aisladas de S. pneumoniae a penicilina y ceftriaxona con la prueba de epsilometría (E-test). Los serotipos de las cepas se determinaron con la reacción de Quellung. RESULTADOS: La incidencia anual de ENI disminuyó significativamente de 7,71 (intervalo de confianza [IC] del 95%: de 1,99 a 13,4) a 1,58 (IC del 95%: de 0,6 a 3,77; reducción del riesgo relativo=-79,5; p=0,006) cada 100 000 habitantes de ≤ 5 años de edad sin enfermedad preexistente. Durante todo el período del estudio, los serotipos en la VCN7 y en la VCN13 representaron el 27,8% y el 63,8% de las cepas aisladas, respectivamente. Los serotipos en la VCN13 correspondían al 81,8% de los casos de ENI en la era previa a la introducción de esta vacuna, y disminuyeron al 56% en los cuatro años posteriores. Las tasas de resistencia a penicilina y ceftriaxona (en el caso de la meningitis) fueron del 48,5% y el 9,1%, respectivamente. CONCLUSIONES: Este estudio observó una disminución significativa en la incidencia de ENI después de la introducción de la VCN13.

[Invasive pneumococcal disease in two non-vaccinated pediatric cases: pleural empyema and bacteremia]. / Asisiz iki çocuk olguda invazif pnömokokal hastalik: Plevral ampiyem ve bakteriyemi.

Streptococcus pneumoniae, a gram-positive diplococcus, is the causative agent of invasive pneumococcal diseases (IPDs) characterized by severe infections such as bacteraemia, sepsis and meningitis. S.pneumoniae and IPDs are situated in the focus of the vaccine studies because of being encompassed of a significant burden of disease in the world, severe mortality and morbidities, and location in vaccine-preventable diseases group. Although S.pneumoniae has more than 90 defined serotypes, certain serotypes are often identified as the cause of IPDs. Individuals with comorbid and chronic diseases, primary or secondary immune deficiencies, and <2 years or >65 years of age are at increased risk for IPDs. Currently, a 23-valent polysaccharide vaccine and also 7, 10 and 13 valent pneumococcal conjugated vaccines (PCV) have been produced for pneumococci. Phase studies of protein based vaccines, which will provide protection independent of serotypes, and 15-valent pneumococcal conjugated vaccine are still ongoing. In Turkey, in November 2008 PCV7 and in April 2011 PCV13 have been implemented in the national immunization program. First case of the pneumococcal unvaccinated cases presented in this report was a 6-year-old girl patient with pneumonia and pleural empyema due to S.pneumoniae serotype 1, without any underlying risk factors. The other case is a 52-days-old male patient, who had a history of pneumococcal septicemia in the newborn period and was followed for bacteremia associated S.pneumoniae serotype 12B and diagnosed as complement deficiency on follow-up. S.pneumoniae serotype 1 is within serotypes covered by 10 and 13 valent pneumococcal conjugate vaccines and pneumococcal polysaccharide vaccine that are in use today, and is a highly invasive strain often isolated in pneumococcal lobar pneumonia and empyema. S.pneumoniae serotype 12B is a non-vaccine serotype not included in any of conjugate and polysaccharide vaccines, and usually obtained in respiratory infections and nasopharyngeal carriage studies. The first case of this report was presented because of an IPD with a serotype included in PCV13 implemented in the routine childhood vaccination schedule and to give an idea about pneumococcal strains circulating in the community. The second case was discussed to draw attention for the evaluation of immune deficiencies and other risk factors in recurrent infections with encapsulated bacteria such as pneumococci. Pneumococcal conjugate vaccines contribute the public immunity with the reduction of vaccine-type pneumococcal nasopharyngeal carriage, IPD incidence, and IPD associated morbidity and mortality especially in young children, at the same time cause a decrease in the prevalence of antibiotic-resistant infections. Application of the pneumococcal conjugate vaccines covering the whole society is important, according to all these important results.

[A pediatric case of pneumococcal meningitis due to Streptococcus pneumoniae serotype 35F]. / Streptococcus pneumoniae serotip 35F'ye bagli pnömokokal menenjitli bir çocuk olgu.

Pneumococci are one of the most common causes of bacterial meningitis in children. It's also responsible for the other invasive pneumococcal diseases (IPD) including bacteremia and pneumonia worldwide. Unvaccinated children are more prone to IPD. Although IPD tend to have a higher prevalence under 2 years of age and in children with primary/secondary immunodeficiencies, and various predisposing factors, older age groups with no underlying diseases also experience IPD. In this report, a pediatric case diagnosed with meningitis due to Streptococcus pneumoniae serotype 35F with no underlying condition and no history of pneumococcal vaccination was presented. An 11-year-old male patient was admitted to the hospital with the complaints of high (39.4°C) fever, headache, vomiting and sleepiness. On the basis of findings from physical examination and laboratory results, the patient was prediagnosed as bacterial meningitis and empirical ceftriaxone and vancomycin therapy was initiated. The cerebrospinal fluid culture of the patient yielded penicillin-susceptible pneumococci and the isolate was identified as serotype 35F by quellung reaction. Vancomycin treatment discontinued depending on the culture result, and the patient fully recovered with 14-days of ceftriaxone therapy without any complications during his follow-ups. Although effective antibiotics are available for IPD, vaccination is indispensable considering the high mortality rates. Seven serotypes (1, 5, 6A, 6B, 14, 19F, 23F) which are currently included in the vaccine, were the most common serotypes related to IPD globally. After mass infant vaccination has been introduced, invasive pneumococcal diseases due to the vaccine serotypes have tended to decrease in both vaccinated young children and non-vaccinated age groups due to herd immunity. Nevertheless, non-vaccine serotypes (NVTs) have emerged as the agents of IPD as a result of serotype replacement. 13-valent pneumococcal conjugate vaccine (PCV13) was introduced in April, 2011 nationwide in our country. This case report was about a patient who had developed meningitis after the introduction of PCV13. There has been no data evaluating the pneumococcal serotype distribution after PCV13 in our country yet. On February, 2013, the Advisory Committee on Immunization Practices (ACIP) recommended routine use of PCV13 for children aged 6-18 years with underlying disease conditions. However, there is no recommendation for children with no underlying diseases in this age group. Vaccination can be extended for otherwise healthy children older than 6 years of age because of increasing trends in incidence of IPD both with vaccine and NVTs like serotype 35F. Recent studies have indicated the emergence of serotype 35F as a cause of IPD in children over 6 years of age and there have been also reports of IPD cases with 35F after the introduction of PCV13. Although serotype 35F is not yet a well-known serotype causing IPD, it might probably gain importance owing to its increasing frequency and virulence and might attract attention to be considered for inclusion in the future pneumococcal vaccines.

Nasopharyngeal carriage of Streptococcus pneumoniae in healthy Turkish children after the addition of PCV7 to the national vaccine schedule.

UNLABELLED: The aim of this study was to determine serotype distribution and investigate antimicrobial resistance patterns of Streptococcus pneumoniae in healthy Turkish children in the era of community-wide pneumococcal conjugate vaccine (PCV7). The study was conducted on 1,101 healthy children less than 18 years of age. Specimens were collected with nasopharyngeal swabs between April 2011 and June 2011. Penicillin and ceftriaxone susceptibilities were determined by E-test according to the 2008 Clinical Laboratory Standards Institute, and serotypes of the isolates were determined by Quellung reaction. The nasopharyngeal pneumococcal carriage rate was 21.9 % (241/1,101). Using the meningitis criteria of minimum inhibitory concentration values, 73 % of the isolates were resistant to penicillin and 47.7 % of them were resistant to ceftriaxone. Half of all pneumococcal isolates were serotyped as 19F (15.2 %), 6A (15.2 %), 23F (10.3 %), and 6B (9.3 %) and surprisingly, no serotype 19A was isolated. Serotype coverage rates of PCV7 and non-PCV7 were 46.2 and 53.8 %, respectively. The most common penicillin- and ceftriaxone-resistant serotypes were 6A, 6B, 14, 19F, and 23F. Penicillin- and ceftriaxone-resistant isolates were more prevalent in serotypes covered by PCV7 than the non-PCV7 serotypes. CONCLUSION: After the community-wide PCV7 vaccination, more non-PCV7 serotypes were isolated from the carriers compared to the time before PCV7 was used especially the serotype 6A, and the antimicrobial resistance of pneumococci was significantly increased.

[An Early Neonatal Sepsis Case Due to Streptococcus pneumoniae Serotype 7F]. / Streptococcus pneumoniae Serotip 7F'ye Bagli Erken Neonatal Sepsis Olgusu.

Streptococcus pneumoniae infections which are relatively unusual in the neonates, are associated with substantial morbidity and mortality. Although S.pneumoniae has more than 90 serotypes, invasive disease is more common with certain serotypes. Pneumococcal polysaccharide and conjugate vaccines are developed against the serotypes that cause invasive disease. Until now invasive pneumococcal diseases have been defined due to various serotypes in the neonatal period. Here we present a neonate with an invasive pneumococcal disease due to S.pneumoniae serotype 7F which has not yet been reported in the literature. A 3180 g infant was born to a healthy 31 year old primigravida by normal vaginal delivery at 39 weeks gestation. Since the infant developed uneasiness, decreased sucking reflex and respiratory difficulty at postnatal 10th hour, he was admitted to the neonatal intensive care unit. Upon admission to intensive care unit mechanical ventilation, fluid replacement and empirical penicillin G and netilmycin had been initiated since the patient had hypotension, hypothermia and tachycardia. The infant later developed severe metabolic acidosis and had anemia, leukocytosis and high CRP values. CSF culture revealed no bacterial growth, however, S.pneumoniae was grown in the blood culture. The isolate was identified as serotype 7F by capsule swelling reaction. The vaginal specimen taken from the mother for the determination of the infectious foci revealed no pathogen growth. The patient's general condition improved after the second day of supportive treatment and antibiotherapy. Since the isolate was penicillin susceptible, the treatment continued with penicillin G and netilmycin for 14 days and the case was discharged with cure. This case was presented to emphasize that S.pneumoniae serotype 7F might lead to invasive disease not only during childhood but also during early neonatal period.

Risk factors for nasopharyngeal carriage of Streptococcus pneumoniae in healthy Turkish children after the addition of heptavalent pneumococcal conjugate vaccine (PCV7) to the national vaccine schedule.

The purpose of this study was to investigate the effects of pneumococcal conjugate vaccine (PCV7) on nasopharyngeal (NP) carriage rates of Streptococcus pneumoniae in healthy Turkish children. The study was conducted on 1101 healthy Turkish children between 1 month and 18 years of age. The median and mean ages of the children were 25 months (1 month-18 years) and 45.7±49.6 months, respectively. S. pneumoniae was isolated in 241/1101 (21.9%) children included in the study. According to multivariate analysis, being <5 years of age, presence of a child attending a daycare center, recovery from respiratory infection within the last month, low income level of the family, and presence of more children in the family were found to be the risk factors for the NP pneumococcal carriage. The carriage rate of NP pneumococci in healthy children was not influenced by PCV7 in Turkey.

The clinical characteristics and treatment of pertussis patients in a tertiary center over a four-year period.

We performed a retrospective study of pertussis patients seen during the period July 2007-December 2011. All patients were microbiologically confirmed through polymerase chain reaction (PCR) or culture. Thirty one patients with positive Bordetella spp. culture or PCR were identified, with a median age of two months. Seventeen patients had received no pertussis vaccination. The most frequent symptoms were paroxysmal cough (100%) and cyanosis (87.1%). The mean duration of the symptoms prior to admission was 12.8 ± 8.0 days. Leukocytosis and thrombocytosis were detected in 64.3% and 67.9% of the 28 hospitalized patients, respectively. Erythromycin had been given to eight patients and clarithromycin to 23 patients. The mean hospital stay was 9.5 ± 5.5 days (range: 3 to 28 days). No patients were readmitted or died. We believe that patients with characteristic paroxysmal cough with cyanosis but no fever should alert clinicians to the possibility of pertussis, and they should be treated without delay. PCR assay provided additional benefit in the diagnoses of the study patients.

[Development and optimization of an in-house PCR method for molecular diagnosis of pertussis]. / Bogmacanin Moleküler Tanisi Için Laboratuvar Yapimi Bir PCR Yönteminin Gelistirilmesi ve Optimizasyonu.

Pertussis (whooping cough), caused by Bordetella pertussis is a severe, acute contagious disease of the respiratory system and it affects mostly children and also susceptible individuals of all ages. Although the conventional culture method used for diagnosis is highly specific, it has a lower sensitivity. Therefore, there is a need for a sensitive, specific and rapid method for diagnosis of pertussis. Polymerase chain reaction (PCR), introduced recently as a new approach for diagnosis of pertussis, has been shown to be more sensitive than culture method. Pertussis toxin gene (ptxA-Pr), insertion sequence genes (IS481 and IS1001), adenylate cyclase genes and structural porin and flagellin genes were chosen as targets for PCR, in different studies. This study aimed to develop and optimize a diagnostic inhouse PCR method by using primers specific for ptxA-Pr and IS481 gene regions. An in-house PCR method was developed by using primer pairs of PTp1/PTp2 specific for ptxA-Pr gene and PIp1/PIp2 specific for IS481 gene and DNAs of various bacterial reference strains. Throat samples obtained from 45 healthy individuals and B.pertussis reference strain with decreasing concentrations were mixed to constitute a group of "representative clinical samples" and used to test and optimize sensitivity and specificity of the method. The in-house PCR with PTp1/PTp2 primers showed a very high specificity but a low sensitivity with a value of 34.4 cfu/Rm (colony forming unit/reaction mixture). Whereas, the inhouse PCR with PIp1/PIp2 primers exhibited a low specificity due to cross-reactivity with B. Pertussis and B.bronchiseptica but much higher sensitivity with a value of 1.12 cfu/Rm. The experiments performed with the representative clinical samples yielded similar results. Simultaneously applied cultivation studies indicated the detection limit of the PCR method as 2 x 103 cfu/ml. Based on our results, the PCR targeting IS481 gene had high sensitivity while the PCR targeting ptxA-Pr gene had high specificity. It was concluded that, PCR method targeting the IS481 gene might be used for pre-diagnosis and then PCR for ptxA-Pr gene might be applied for the confirmation of B.pertussis in the molecular diagnosis of pertussis.