Establishment and Modification of Ninety-seven Pneumococcal Serotyping Assays Based on Quantitative Real-time Polymerase Chain Reaction / 生物医学与环境科学（英文）

OBJECTIVE@#To establish and modify quantitative real-time polymerase chain reaction (qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.@*METHODS@#A database of capsular polysaccharide ( cps) loci sequences was generated, covering 97 pneumococcal serotypes. Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs. A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR, while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay. A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.@*RESULTS@#A total of 97 pneumococcal serotyping assays based on qPCR were established and modified, which included 64 serotypes previously reported as well as an additional 33 serotypes. Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system. A total of 97 pneumococcal serotypes, which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups, could not be identified as individual serotypes. The sensitivity of qPCR assays based on 27 target sequences was 1-100 copies/µL. The specificity of the qPCR assays was 100%, which were tested by a panel of 90 serotypes of the pneumococcal reference strains.@*CONCLUSION@#A total of 27 novel qPCR assays were established and modified to analyze 97 pneumococcal serotypes.

Dose-response relationship between age and Streptococcus pneumoniae vaccination coverage in kindergarten children / 中国当代儿科杂志

OBJECTIVES@#To investigate the potential relationship between age and Streptococcus pneumoniae vaccination coverage in kindergarten children, and to provide a basis for guiding vaccination and developing new protein vaccines.@*METHODS@#The stratified cluster random sampling method was used to select 1 830 healthy children from six kindergartens in Shunde District, Foshan City, China, and nasopharyngeal swabs were collected for the isolation and identification of Streptococcus pneumoniae. The logistic regression model based on restricted cubic spline was used to analyze the dose-response relationship between age and Streptococcus pneumoniae vaccination coverage.@*RESULTS@#The rate of nasal Streptococcus pneumoniae carriage was 22.46% (411/1 830) among the kindergarten children, with the predominant serotypes of 6B, 19F, 15A, 23A, 34, and 23F. The coverage rates of 10-valent pneumococcal conjugate vaccine (PCV10) and 13-valent pneumococcal conjugate vaccine (PCV13) were 53.0% and 57.9%, respectively, and there was a significant non-linear dose-response relationship between age and the coverage rates of PCV10 and PCV13 (P<0.05), with a higher coverage rate of PCV10 (88.0%) and PCV13 (91.1%) in the children aged 2 years. There was a significant non-linear dose-response relationship between age and the coverage rates of pilus islet 1 (PI-1) and pilus islet 2 (PI-2) (P<0.05), with a lower vaccination coverage rate for PI-1 (37.7%) and PI-2 (16.1%). The coverage rates of PI-1 (13.0%-58.5%) and PI-2 (6.0%-29.4%) were lower in all age groups. The virulence genes lytA (99.5%) and ply (99.0%) associated with candidate protein vaccines showed higher vaccination coverage rates.@*CONCLUSIONS@#There is a significant non-linear dose-response relationship between the age of kindergarten children and the coverage rates of PCV10 and PCV13 serotypes, and kindergarten children aged 2 years have a relatively high coverage rate of PCV. The high prevalence of the virulence genes lytA and ply shows that they are expected to become candidate virulence factors for the development of a new generation of recombinant protein vaccines.

Clinical and microbiological characteristics of community-acquired pneumonia associated with Streptococcus pneumoniae in adult patients in Mexico / Características clínicas y microbiológicas de la neumonía adquirida en la comunidad asociada con Streptococcus pneumoniae en pacientes adultos en México

The aim of this study was to assess the risk factors and clinical and microbiological characteristics of community-acquired pneumonia (CAP) in adult patients in Mexico. Streptococcus pneumoniae classified as the causative agent of CAP in adult patients and patients with invasive S. pneumoniae isolates presented to three tertiary teaching hospitals during the 15-year study period were selected. Serotyping and susceptibility testing were performed for all included isolates. Clinical and demographic data were recorded. A total of 96 patients infected with S. pneumoniae (71 with CAP, 25 with invasive disease) were included. The CAP group involved more males (74.6%) than the invasive disease group (p = 0.03). Head trauma was more common in the CAP group (21.1%) than in the invasive disease group (4.0%; p = 0.03). The most prevalent serotype was 19A, followed by serotypes 3 and 23F. After the introduction of the heptavalent conjugated pneumococcal vaccine (PCV7), the prevalence of included serotypes declined significantly; no such change was found after the introduction of the PCV13 vaccine, including in the prevalence of serotype 19A. Susceptibility to all antimicrobials tested except vancomycin declined over the study period. In conclusion, head trauma was the most common comorbidity in the CAP group. The most prevalent serotype was 19A. Decreased susceptibility to most antimicrobials tested was observed.

El objetivo de este estudio fue evaluar los factores de riesgo y las características clínicas y microbiológicas de la neumonía adquirida en la comunidad (NAC) en pacientes adultos en México. Se seleccionaron pacientes adultos con NAC con Streptococcus pneumoniae como agente causal y pacientes con aislamientos invasivos de S. pneumoniae que concurrieron a tres hospitales de enseñanza de tercer nivel durante el período de estudio de 15 anos (2000-2015). Se realizaron pruebas de serotipificación y sensibilidad con todos los aislados incluidos. Se colectaron los datos clínicos y demográficos. Se incluyeron en total 96 pacientes infectados con S. pneumoniae (71 con NAC y 25 con enfermedad invasiva). El grupo con NAC incluía más varones (74,6%) que el grupo de enfermedad invasiva (p = 0,03). El traumatismo craneoencefálico fue más frecuente en el grupo NAC (21,1%) queen el grupo con enfermedad invasiva (4,0%; p = 0,03). El serotipo más frecuente fue 19A, seguido de los serotipos 3 y 23F. Después de la introducción de la vacuna antineumocócica conjugada heptavalente (PCV7), la prevalencia de los serotipos incluidos en aquella disminuyó significativamente; no sucedió lo mismo después de la introducción de la PCV13, incluso en relación con la prevalencia del serotipo 19A. La sensibilidad a todos los antimicrobianos evaluados, excepto la vancomicina, disminuyó durante el período de estudio. En conclusión, el traumatismo craneoencefálico fue la comorbilidad más frecuente en el grupo con NAC. El serotipo más frecuente fue el 19A, y se observó disminución de la sensibilidad a la mayoría de los antimicrobianos probados a lo largo del período considerado.

Antimicrobial susceptibility and fluctuations in clonal complexes of serogroup 6 Streptococcus pneumoniae isolates collected from children in Beijing, China, between 1997 and 2016

ABSTRACT This study examined the antimicrobial susceptibility patterns and clonal complex (CC) characteristics of serogroup 6 Streptococcus pneumoniae isolates collected from children in Beijing, China, between 1997 and 2016. Serotypes were determined using the Quellung reaction, and the antimicrobial susceptibility profiles of the isolates were determined using the disc-diffusion method or by E-test. Sequence types (STs) were assigned based on multilocus sequence typing. A total of 250 isolates were examined, with 55.2%, 30.0%, 12.8%, and 2.0% of isolates identified as serotypes 6A, 6B, 6C, and 6D, respectively. All of the isolates were susceptible to levofloxacin and vancomycin, and the non-suceptibitility rate to penicillin was 41.6%. Eighty-two distinct STs, assigned to 13 CCs and 28 singletons, were identified. CC982 was the most prevalent CC amongst serotype 6A isolates (34%), followed by CC9789 and CC3173. Amongst serotype 6B isolates, CC90 and CC4542 were the most common, accounting for 25.3% and 14.7% of isolates respectively. Over the study period, the prevalence of CC982, CC4542, and CC4536 isolates showing susceptibility to penicillin and cefuroxime decreased, and the proportion of CC3173, CC9789, CC855, and CC902 isolates showing non-susceptibility to these two antibiotics increased.

Resistencia antibiótica y distribución de serotipos en cepas neumocócicas invasivas en adultos hospitalizados en Lima, Perú / Antibiotic resistance and distribution of serotypes of invasive pneumococcal strains isolated from hospitalized adults in Lima, Peru

RESUMEN Objetivos. Describir las características clínicas, resistencia antibiótica y distribución de serotipos de cepas causantes de enfermedad neumocócica invasiva (ENI) en adultos. Materiales y métodos. Estudio tipo serie de casos. Se recolectaron cepas de neumococo de pacientes adultos hospitalizados con ENI en cinco hospitales nacionales y dos laboratorios de Lima durante los años 2009-2011. Resultados. Se estudiaron datos de 43 pacientes con ENI, el 58,2% fueron mayores de 60 años. Los diagnósticos fueron neumonía 39,5%, meningitis 30,2%, bacteriemia 13,9%, peritonitis 11,6%, artritis séptica 4,8%. El porcentaje de fallecidos fue 28,9%, de los cuales el 72,7% fueron mayores de 60 años. Las cepas de neumococo presentaron la siguiente resistencia: penicilina 0% en cepas no meningitis y 30,8% en cepas meningitis; ceftriaxona 4,5% y 16,7% de resistencia intermedia en cepas no meningitis y cepas meningitis respectivamente; 69% a trimetoprim/sulfametoxazol y 35,7% a eritromicina. Los serotipos más comunes fueron 19F, 23F, 6B, 14 y 6C. El porcentaje de cepas vacunales fue 44,2% para la vacuna conjugada siete-valente (PCV7) y para la PCV10, 51,2% para PCV13 y 60,4% para la vacuna polisacárida veintitrés-valente (PPV23). Conclusiones. El neumococo es un patógeno relevante en adultos, en especial en los adultos mayores, debido a su elevada mortalidad.

ABSTRACT Objectives. To describe the clinical characteristics, antibiotic resistance, and distribution of serotypes of bacterial strains that cause invasive pneumococcal disease (IPD) in adults. Materials and methods. Case series. Pneumococcal strains were isolated from 2009 to 2011 from hospitalized adult patients with IPD in five hospitals and two laboratories located in Lima. Results. The analysis of data from 43 patients with IPD indicated that 58.2% were older than 60 years. The most common complications were pneumonia (39.5%), meningitis (30.2%), bacteremia (13.9%), peritonitis (11.6%), and septic arthritis (4.8%). The mortality rate was 28.9%, and 72.7% of cases involved patients older than 60 years. The pneumococcal strains were resistant to the following antibiotics: penicillin, 0% and 30.8% in non-meningitis and meningitis strains, respectively; ceftriaxone, 4.5% and 16.7% in non-meningitis and meningitis strains, respectively; trimethoprim/sulfamethoxazole, 69.0%; and erythromycin, 35.7%. The most common serotypes were 19F, 23F, 6B, 14, and 6C. The percentage of vaccine strains was 44.2% for the 7-valent conjugate pneumococcal vaccine (PCV7) and PCV10, 51.2% for PCV13, and 60.4% for the 23-valent polysaccharide vaccine (PPV23). Conclusions. Pneumococcus is an important pathogen in adults, particularly in older adults, owing to its high mortality rate.

Molecular characterization of non-vaccine Streptococcus pneumoniae serotypes 11A, 15 B/C and 23A recovered from invasive isolates in Colombia / Caracterización molecular de los serotipos no vacunales 11A, 15 B/C y 23A de Streptococcus pneumoniae recuperados de aislamientos invasivos en Colombia

Resumen Introduction: A total of 192 invasive Streptococcus pneumoniae isolates, from serotypes 11A, 15B/C and 23A (not included in the conjugated vaccines), were collected in Colombia between 1994 and 2014 as part of the activities of the Network surveillance system for the causative agents of pneumonia and meningitis (SIREVA II). Objective: To determine the molecular characteristics ofinvasive S. pneumoniaeisolates from serotypes 11A, 15B/C and 23A in Colombia from 1994 to 2014. Materials and methods: The molecular characterization of the isolates was carried out through Pulse-Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST). Results: Serotype 11A showed one clonal group represented by ST62. Serotype 15B/C was composed of three groups associated with Netherlands15B-37 ST199 (28.75%), ST8495 (18.75%), and SLV (Single-Locus Variant) of ST193 (21.25%). Isolates from serotype 23A were gathered in three clonal groups, with70.21% closely related toST42, 17.02% to Colombia23F-ST338, and6.38% to Netherlands15B-37 ST199. Conclusion: Clones Colombia23F-ST338 andNetherlands15B-ST199 covered more serotypes than those previously found by other authors, including serotype 23A. These analyses reveal the importance of capsular switching in the spreading of successful clones among non-vaccine serotypes causing invasive pneumococcal disease.

Abstract Introducción. En Colombia se recolectaron 192 aislamientos invasivos de Streptococcus pneumoniae de los serotipos 11A, 15B/C y 23A (no incluidos en las vacunas conjugadas) entre 1994 y 2014, como parte de las actividades del Sistema de Redes de Vigilancia de los Agentes Responsables de Neumonías y MeningitisBacterianas (SIREVA II). Objetivo. Determinar las características moleculares de aislamientosinvasivos de los serotipos11A, 15B/C y 23A de S. pneumoniae recolectados en Colombia entre 1994 y 2014. Materiales y métodos. La caracterización molecular de los aislamientos se hizo medianteelectroforesis en gel de campo pulsado (Pulse-Field Gel Electrophoresis, PFGE) y por tipificación de secuencias multilocus (Multilocus Sequence Typing, MLST). Resultados. El serotipo 11A mostró un grupo clonal representadopor el ST62, en tanto que el serotipo15B/C se distribuyó en tres grupos asociados conlos clones Netherlands15B-37 ST199 (28,75 %), ST8495 (18,75 %) y SLV (variante en un solo locus) de ST193 (21,25 %). Los aislamientos con serotipo 23A se agruparon en tres gruposclonales; 70,21 % de ellos estaban estrechamente relacionadoscon elST42, 17,02 % con elColombia23F-ST338, y 6,38 % con el Netherlands15B-37 ST199. Conclusión. Los clones Colombia23F-ST338 y Netherlands15B-ST199 encontrados en este estudio abarcaronmás serotipos de los reportados previamente por otros autores, incluido el serotipo23A. Estos análisis revelan laimportancia de la conmutación(switching) capsular en la expansión de clones exitosos entre los serotipos no vacunales como causa de enfermedad invasiva neumocócica.

Comparison of culture and PCR methods in the diagnosis of bacterial meningitis

Abstract Our aim in this study is to compare the standard culture method with the multiplex PCR and the Speed-Oligo® Bacterial Meningitis Test (SO-BMT) – a hybridization-based molecular test method – during the CSF examination of the patients with the pre-diagnosis of acute bacterial meningitis. For the purposes of this study, patients with acute bacterial meningitis treated at the Dicle University Medical Faculty Hospital, Infectious Diseases and Clinical Microbiology Clinic between December 2009 and April 2012 were retrospectively evaluated. The diagnosis of bacterial meningitis was made based on the clinical findings, laboratory test anomalies, CSF analysis results, and the radiological images. Growth was observed in the CSF cultures of 10 out of the 57 patients included in the study (17.5%) and Streptococcus pneumoniae was isolated in all of them. The CSF samples of 34 patients (59.6%) were positive according to the SO-BMT and S. pneumoniae was detected in 33 of the samples (97.05%), while Neisseria meningitidis was found in 1 sample (2.95%). In a total of 10 patients, S. pneumoniae was both isolated in the CSF culture and detected in the SO-BMT. The culture and the SO-BMT were negative in 23 of the CSF samples. There was no sample in which the CSF culture was positive although the SO-BMT was negative. While SO-BMT seems to be a more efficient method than bacterial culturing to determine the pathogens that most commonly cause bacterial meningitis in adults, further studies conducted on larger populations are needed in order to assess its efficiency and uses.

The virulence of Streptococcus pneumoniae partially depends on dprA

Abstract Streptococcus pneumoniae is one of the most frequent opportunistic pathogens worldwide. DNA processing protein A (DprA) is an important factor involved in bacterial uptake and DNA integration into bacterial genome, but its role in S. pneumoniae virulence remains unclear. The aim of this study was to characterize the effects of the pneumococcal dprA gene on the pathogenesis of S. pneumoniae. To construct a dprA-deficient pneumococcal strain, the dprA gene of the S. pneumoniae strain D39 was inactivated. The virulence of this dprA-deficient strain, designated ΔD39, was compared with that of the wild-type strain by evaluating their respective capabilities to adhere to human pulmonary epithelial cells (PEC-A549) and by analyzing their choline-binding protein expression levels. In addition, the expression profiles of genes associated with virulence and host survival assays were also conducted with the mutant and the wild-type strain. Our results indicate that the capability of ΔD39 to adhere to the PEC-A549 airway cells was significantly lower (p < 0.01) compared with D39. Additionally, the 100-KD choline-binding protein was not detected in ΔD39. The addition of competence-stimulating peptide (CSP) lead to a significantly reduction of psaA mRNA expression in the dprA-deficient mutant and an increased level of psaA transcripts in the wild-type strain (p < 0.01). The median survival time of mice intraperitoneally infected with ΔD39 was significantly higher (p < 0.01) than that of mice infected with D39. The results of this study suggest that DprA has a significant effect on virulence characteristics of S. pneumoniae by influencing the expression of choline-binding protein and PsaA.

Comparison of PCR-based methods for the simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical samples

Abstract Background Several in-house PCR-based assays have been described for the detection of bacterial meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from clinical samples. PCR-based methods targeting different bacterial genes are frequently used by different laboratories worldwide, but no standard method has ever been established. The aim of our study was to compare different in-house and a commercial PCR-based tests for the detection of bacterial pathogens causing meningitis and invasive disease in humans. Methods A total of 110 isolates and 134 clinical samples (99 cerebrospinal fluid and 35 blood samples) collected from suspected cases of invasive disease were analyzed. Specific sets of primers frequently used for PCR-diagnosis of the three pathogens were used and compared with the results achieved using the multiplex approach described here. Several different gene targets were used for each microorganism, namely ctrA, crgA and nspA for N. meningitidis, ply for S. pneumoniae, P6 and bexA for H. influenzae. Results All used methods were fast, specific and sensitive, while some of the targets used for the in-house PCR assay detected lower concentrations of genomic DNA than the commercial method. An additional PCR reaction is described for the differentiation of capsulated and non-capsulated H. influenzae strains, the while commercial method only detects capsulated strains. Conclusions The in-house PCR methods here compared showed to be rapid, sensitive, highly specific, and cheaper than commercial methods. The in-house PCR methods could be easily adopted by public laboratories of developing countries for diagnostic purposes. The best results were achieved using primers targeting the genes nspA, ply, and P6 which were able to detect the lowest DNA concentrations for each specific target.

Meningitis neumocóccica por serotipo 19 A, de curso fatal: ¿Un caso prevenible? / Fatal pneumococcal meningitis due to serotype 19 A: A preventable case?

Invasive pneumococcal disease (IPD) remains as an important cause of morbidity in the world and in our country, while in Chile the incidence has decreased after the incorporation of the 10 valent pneumococcal conju-gate vaccine, in the routine infant inmunization schedule (EPI). One of the expected effects of the program after vaccination with 10-valent pneumococcal vaccine is the likely replacement serotype phenomenon that means the presence of ENI caused by serotypes not included in the vaccine. In this context, we present the case of a child with pneumococcal meningitis caused by serotype 19 A of fatal course. The occurrence of ENI in a later stage of pneumococcal vaccine incorporation in Chile reinforces the importance of active surveillance, in order to know in detail the impact of vaccination, distribution of circulating serotypes and their correlation with the different clinical disease and their severity.

La enfermedad neumocóccica invasora (ENI) sigue siendo una causa importante de morbilidad en el mundo y en nuestro país, si bien en Chile la incidencia ha disminuido luego de la incorporación de la vacuna neumocóccica conjugada 10-valente al Programa Nacional de Inmunizaciones (PNI). Uno de los efectos esperables luego de la vacunación programática con la vacuna antineumocóccica 10-valente es el probable fenómeno de reemplazo, que corresponde a la presencia de ENI por serotipos no incluidos en la vacuna. En este contexto, se presenta el caso de un pre-escolar con meningitis neumocóccica causada por el serotipo 19 A, de curso fatal. La presencia de casos de ENI en una etapa posterior a la implementación de la vacuna anti-neumocóccica en el PNI de Chile, demuestra la importancia de realizar una vigilancia activa, con el objetivo de conocer en forma detallada el impacto de la vacunación, la distribución de los serotipos circulantes y su correlación con los diferentes cuadros clínicos y su evolución.

Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae causing noninvasive diseases in a Children's Hospital, Shanghai

Background: Streptococcus pneumoniae, which cause noninvasive pneumococcal diseases, severely impair children's health. This study analyzed serotype distribution and antimicrobial resistance of S. pneumoniae from January 2012 to December 2012 in a Children's Hospital, Shanghai. Methods: A total of 328 pneumococcal isolates were serotyped by multiplex sequential PCR and/or capsule-quellung reaction. The minimum inhibitory concentrations for 11 antimi- crobial agents were determined by broth microdilution method. Results: Among 328 strains, 19F (36.3%), 19A (13.4%), 6A (11.9%), 23F (11.0%), 14 (5.8%), 6B (5.2%), and 15B/C (4.3%) were the most common serotypes. The coverage rates of 7-, 10-, and 13-valent conjugate vaccines (PCV7, PCV10, and PCV13) were 58.2%, 58.2%, and 84.1%, respectively. Out of the isolates, 26 (7.9%) strains were penicillin resistant. Most of the strains displayed high resistance rate to macrolides (98.5% to erythromycin, 97.9% to azithromycin, and 97.0% to clindamycin). Conclusions: The potential coverage of PCV13 is higher than PCV7 and PCV10 because of the emergence of 19A and there should be long-term and systematic surveillance for non-vaccine serotypes. .

Distrofia muscular facioescapulohumeral en Chile: presentación de serie en hospital de referencia terciario / Facioscapulohumeral muscular dystrophy: Report of seven patients

Background: Facioscapulohumeral muscular dystrophy is the third most common muscular dystrophy with an estimated prevalence of 1 per 20.000 and a normal life expectancy in the majority of patients. However, approximately 15% of patients become wheelchair bound in the course of their life. It is a hereditary autosomal dominant disease with high (95%) penetrance by the age of 20, but with variable degree of phenotypic expression even in the same family group. Symptoms frequently start in the second decade of life, with facial and scapular weakness. Aim: To report the clinical features of seven patients with the disease, seen at a public hospital. Material and Methods: Analysis of seven patients with genetic study seen in a public Hospital in Santiago. Results: The age of patients fluctuated from 18 to 61 years and four were females. The mean age at onset of symptoms was 29 years and four had a family history of the disease. The usual presenting complaint was arm or shoulder asymmetric weakness. Four patients had bone pain. Facial involvement was present in four. A genetic study was done in five patients, the other two patients were relatives, confirming the contraction or lower number of repetitions in D4Z4 region. After 12 years of follow up only 2 patients older than 60 years cannot work and one female patients is in a semi dependent state at the age of 30. Conclusions: The clinical workup in the diagnosis and the timely indication of genetic studies are highlighted, to avoid unnecessary and invasive procedures. The variability in the phenotypic expression in a similar genetic defect is discussed and the genetic or epigenetic mechanisms of this muscular dystrophy are described.

Differentiation of Haemonchus placei from Haemonchus contortus by PCR and by morphometrics of adult parasites and third stage larvae / Identificação de Haemonchus placei e Haemonchus contortus por PCR e por análise morfológica de parasitas adultos e larvas infectantes

Molecular and morphological methods were evaluated to distinguish between Haemonchus contortus and Haemonchus placei species. A total of 141 H. contortus and 89 H. placei male adult specimens collected from artificially infected lambs were identified individually by PCR analysis, using a species-specific primer pair. These PCR results were used as gold standard for Haemonchus spp. identification. Haemonchus placei presented higher mean spicule and barb lengths than H. contortus (P<0.05). However, some measurements overlapped. For this reason, a discriminate function did not allow the correct identification of 13 H. contortus and one H. placei specimen. The sheath tail length of the third stage larvae (L3), which comprises the distance between the tip of the larval tail and the end of the sheath tail, were measured. Only three of the 485 H. placei larvae (0.619%) had a sheath tail shorter than 85 µm, while only four of the 500 H. contortus larvae (0.8%) presented a sheath tail longer than 85 µm. The results indicated that 6.09% of the male adult specimens would be misclassified based on the discriminate function, while only 0.71% of infective larvae would be misclassified. Therefore, identification of L3 can be used as the first method to indicate the presence of H. placei and/or H. contortus in a population of domestic ruminants.

Métodos moleculares e morfológicos foram avaliados para a identificação de Haemonchus contortus e Haemonchus placei. No total, 141 H. contortus e 89 H. placei machos adultos, obtidos de cordeiros artificialmente infectados, foram identificados individualmente por PCR com o emprego de um par de “primers” espécie-específico. Esses resultados da análise por PCR foram considerados como padrão para a identificação das espécies de Haemonchus. Haemonchus placei apresentou valores médios de espículos e ganchos superiores aos de H. contortus (P<0,05). Entretanto, houve sobreposição de alguns valores. Por essa razão, a função discriminante não permitiu a identificação correta de 13 exemplares de H. contortus e de um, de H. placei. Foi medida a cauda da bainha de larvas infectantes (L3), que compreende a distância entre a ponta da cauda da larva e a ponta da cauda da bainha. Apenas três das 485 L3 de H. placei (0,619%) apresentaram a cauda da bainha com medida inferior a 85 µm e somente em quatro das 500 L3 de H. contortus (0,8%) essa medida foi superior a 85 µm. Os resultados demonstraram que 6,09% dos machos adultos seriam identificados erroneamente com base na função discriminante, enquanto a identificação incorreta de L3 seria de apenas 0,71%. Portanto, a identificação de L3 pode ser utilizada como método inicial para indicar a presença de H. placei e/ou H. contortus em uma população de ruminantes domésticos.

Macrolide resistance in Streptococcus pneumoniae isolated from Argentinian pediatric patients suffering from acute otitis media / Macrolide resistance in Streptococcus pneumoniae isolated from Argentinian pediatric patients suffering from acute otitis media.

Macrolide-resistant Streptococcus pneumoniae emerged in Argentina in 1995, representing 26

of invasive infection isolates in children under 5 years old. The objectives of this study were to describe the prevalence of ermB and mefA genes in macrolide-resistant S. pneumoniae isolates from acute otitis media (AOM) and to determine their genetic relatedness. Between May 2009 and August 2010, 126 S. pneumoniae isolates from 324 otherwise healthy children with a first episode of AOM were included. Twenty six of these isolates (20.6

) were resistant to erythromycin. Most frequent serotypes were: 14 (46.2

) and 9V (7.7

) carried the mefA gene, 5 (19.2

) have the ermB gene, and 1 (3.9

) both ermB + mefA. Ten clonal types were identified, mostly related to Sweden(15A)-25/ST782 (SLV63), CloneB(6A)/ST473 and England(14)-9/ ST9. This is the first study assessing the mechanisms of macrolide resistance in pneumococci isolates from pediatric AOM in Argentina and their genetic relatedness.

Molecular characterization of Streptococcus pneumoniae serotype 1 invasive isolates in Colombia / Caracterización molecular de cepas invasoras de Streptococcus pneumoniae serotipo 1 aisladas en Colombia

OBJECTIVE: To determine the genetic relationship between Streptococcus pneumoniae serotype 1 Colombian isolates recovered from invasive disease between 1994 and 2011 and recognized serotype 1 international clones. METHODS: A total of 135 S. pneumoniae serotype 1 isolates with epidemiological and antimicrobial susceptibility data (Clinical and Laboratory Standards Institute, 2012) were studied. The genetic relationship with recognized international clones was established by pulsed-field gel electrophoresis (PFGE) with SmaI restriction enzyme. Multilocus sequence typing (MLST) was standardized to determine the sequence type (ST) in seven isolates representing different clonal groups. Control and reference strain R6, and clones Sweden¹ ST217, Sweden¹ ST304, Sweden¹ ST306, and USA¹ ST615, were used. RESULTS: PFGE revealed that 89.7% of the isolates were associated with Sweden¹ ST306, 3.7% were associated with Sweden¹ ST304, and 6.6% were not clonally related. Using MLST, ST306 was confirmed in six isolates and ST304 in one. CONCLUSIONS: In contrast to Brazil and the United States, where clones Sweden¹ ST304 and ST227 prevail, invasive disease caused by S. pneumoniae serotype 1 in Colombia is principally associated with the dispersion of isolates related to clone Sweden¹ ST306.

OBJETIVO: Determinar la relación genética entre las cepas de Streptococcus pneumoniae serotipo 1 aisladas en Colombia en casos de enfermedad invasora entre 1994 y 2011 y los clones internacionales reconocidos del serotipo 1. MÉTODOS: Se estudiaron un total de 135 cepas de S. pneumoniae serotipo 1 de las que se tenían datos epidemiológicos y de sensibilidad a los antimicrobianos (Clinical and Laboratory Standards Institute, 2012). Se estableció su relación genética con los clones internacionales reconocidos mediante electroforesis en gel de campo pulsátil (PFGE) utilizando la enzima de restricción SmaI. Se estandarizó la tipificación de secuencias mulitlocus (MLST) para determinar el tipo de secuencia (ST) en siete cepas que representaban diferentes grupos clonales. Se utilizaron la cepa de control y referencia R6 y los clones Sweden¹ ST217, Sweden¹ ST304, Sweden¹ ST306, y USA¹ ST615. RESULTADOS: La PFGE reveló que 89,7% de las cepas se asociaban con Sweden¹ ST306, 3,7% con Sweden¹ ST304, y 6,6% no mostraron relación clonal. Mediante MLST, se confirmó la relación con ST306 en seis cepas y con ST304 en una. CONCLUSIONES: A diferencia de Brasil y Estados Unidos, donde prevalecen los clones Sweden¹ ST304 y ST227, la enfermedad invasora causada por S. pneumoniae serotipo 1 en Colombia se asocia principalmente con la dispersión de cepas relacionadas con el clon Sweden¹ ST306.

Non invasive real-time monitoring of bacterial infection & therapeutic effect of anti-microbials in five mouse models.

Background & objectives: In vivo imaging system has contributed significantly to the understanding of bacterial infection and efficacy of drugs in animal model. We report five rapid, reproducible, and non invasive murine pulmonary infection, skin and soft tissue infection, sepsis, and meningitis models using Xenogen bioluminescent strains and specialized in vivo imaging system (IVIS). Methods: The progression of bacterial infection in different target organs was evaluated by the photon intensity and target organ bacterial counts. Genetically engineered bioluminescent bacterial strains viz. Staphylococcus aureus Xen 8.1, 29 and 31; Streptococcus pneumoniae Xen 9 and 10 and Pseudomonas aeruginosa Xen-5 were used to induce different target organs infection and were validated with commercially available antibiotics. Results: The lower limit of detection of colony forming unit (cfu) was 1.7-log10 whereas the lower limit of detection of relative light unit (RLU) was 4.2-log10. Recovery of live bacteria from different target organs showed that the bioluminescent signal correlated to the live bacterial count. Interpretation & conclusions: This study demonstrated the real time monitoring and non-invasive analysis of progression of infection and pharmacological efficacy of drugs. These models may be useful for pre-clinical discovery of new antibiotics.

Spatial exploration of Streptococcus pneumoniae clonal clustering in São Paulo, Brazil

OBJECTIVES: To examine the spatial distribution of Streptococcus pneumoniae and its clonal patterns collected between 2002 and 2006 in São Paulo, Brazil. METHODS: As part of an observational study in São Paulo city, Brazil, S. pneumoniae isolates routinely cultured from blood, respiratory specimens, or cerebrospinal and other profound fluids were selected. Additionally, only isolates with either penicillin (PEN) intermediate (I) or resistant (R) status on routine antibiogram were included, in order to obtain a higher probability of clonal isolates. A single I/R S. pneumoniae isolate per patient was included and submitted to genotypic determination by pulsed field gel electrophoresis (PFGE). Minimum inhibitory concentrations (MICs) were determined for the isolates by Etest® to PEN and other antimicrobials. Each isolate was geocoded in a digital map. The Kernel function and ratio methods between total isolates vs. clones were used in order to explore possible cluster formations. RESULTS: Seventy-eight (78) S. pneumoniae community isolates from two major outpatient centers in São Paulo, Brazil, were selected from the databank according to their penicillin susceptibility profile, i.e. R or I to penicillin assessed by oxacillin disc diffusion. Of these, 69 were submitted to PFGE, 65 to MIC determination, and 48 to spatial analytical procedures. Preliminary spatial analysis method showed two possible cluster formation located in southwest and southeast regions of the city. CONCLUSION: Further analyses are required for precisely determining the existence of S. pneumoniae clusters and their related risk factors. Apparently there is a specific transmission pattern of S. pneumoniae clones within certain regions and populations. GIS and spatial methods can be applied to better understand epidemiological patterns and to identify target areas for public health interventions.

Análise das cepas de Streptococcus pneumoniae causadores de pneumonia invasiva: sorotipos e sensibilidade aos antimicrobianos / Analysis of invasive pneumonia-causing strains of Streptococcus pneumoniae: serotypes and antimicrobial susceptibility

OBJETIVOS: Identificar os sorotipos de pneumococo mais frequentemente isolados de crianças internadas com pneumonia invasiva, comparar os sorotipos com os incluídos em vacinas conjugadas e analisar sua sensibilidade aos antimicrobianos mais utilizados na faixa etária pediátrica. MÉTODOS: Estudo descritivo, retrospectivo das pneumonias pneumocócicas identificadas em crianças internadas no hospital universitário da Universidade de São Paulo, no período de janeiro de 2003 a outubro de 2008. Os critérios de inclusão foram: faixa etária de 29 dias até 15 anos incompletos com diagnóstico clínico e radiológico de pneumonia e com cultura de sangue e/ou líquido pleural com crescimento de Streptococcus pneumoniae. RESULTADOS: Foram incluídas no estudo 107 crianças. Os sorotipos mais frequentes foram: 14 (36,5 por cento), 1 (16,7 por cento), 5 (14,6 por cento), 6B (6,3 por cento) e 3 (4,2 por cento). A proporção de sorotipos contidos na vacina conjugada heptavalente seria de 53,1 por cento, na vacina 10-valente de 86,5 por cento e na 13-valente seria de 96,9 por cento. De acordo com os padrões do Clinical and Laboratory Standards Institute 2008, 100 cepas (93,5 por cento) de pneumococos foram sensíveis à penicilina (concentração inibitória mínima, CIM < 2 µg/mL), 7 cepas (6,5 por cento) com resistência intermediária (CIM = 4 µg/mL) e nenhuma com resistência (CIM > 8 µg/mL). Verificamos alta taxa de sensibilidade para as cepas testadas para vancomicina, rifampicina, ceftriaxone, clindamicina, cloranfenicol e eritromicina. CONCLUSÕES: Nossos resultados confirmam um expressivo impacto potencial das vacinas conjugadas, principalmente pela 10-valente e 13-valente, sobre os casos de pneumonias invasivas. Os resultados de sensibilidade à penicilina evidenciam que a opção terapêutica de escolha para o tratamento das pneumonias invasivas continua sendo a penicilina.

OBJECTIVES: To identify the most common pneumococcal serotypes in children hospitalized with invasive pneumonia, correlate isolated serotypes with those included in conjugate vaccines, and ascertain the sensitivity of the isolated pneumococcal strains to penicillin and other antibiotics. METHODS: From January 2003 to October 2008, a retrospective study of hospitalized children with a diagnosis of Streptococcus pneumoniae pneumonia was conducted at the university hospital of Universidade de São Paulo. Criteria for inclusion were: age greater than 29 days and less than 15 years, radiological and clinical diagnosis of pneumonia, and isolation of Streptococcus pneumoniae in blood cultures and/or pleural effusion. RESULTS: The study included 107 children. The most common serotypes were 14 (36.5 percent), 1 (16 percent), 5 (14.6 percent), 6B (6.3 percent) and 3 (4.2 percent). The proportion of identified serotypes contained in the heptavalent, 10-valent and 13-valent conjugate vaccines was 53.1, 86.5, and 96.9 percent, respectively. Pneumococcal strains were sensitive to penicillin (minimum inhibitory concentration, MIC < 2 µg/mL) in 100 cases (93.5 percent) and displayed intermediate resistance (MIC = 4 µg/mL) in 7 cases (6.5 percent). No strains were penicillin-resistant (MIC > 8 µg/mL) according to the Clinical and Laboratory Standards Institute 2008 standards. Tested isolates were highly sensitive to vancomycin, rifampicin, ceftriaxone, clindamycin, erythromycin, and chloramphenicol. CONCLUSIONS: Our results confirm a significant potential impact of conjugate vaccines, mainly 10-valent and 13-valent, on invasive pneumonia. Furthermore, susceptibility testing results show that penicillin is still the treatment of choice for invasive pneumonia in our setting.

Circulation of Streptococcus pneumoniae clone Colombia5 ST289 in nine Latin American countries / Circulación de Streptococcus pneumoniae clon Colombia5 ST289 en nueve países de América Latina

OBJECTIVE: To determine genetic relatedness of clone Colombia5 ST289 with invasive Streptococcus pneumoniae serotype 5 isolates recovered in nine Latin American countries. METHODS: Forty-four invasive S. pneumoniae serotype 5 isolates recovered from children under 5 years of age in Bolivia, Chile, Dominican Republic, Ecuador, Nicaragua, Panama, Paraguay, Peru, and Venezuela were studied. Pulsed-field gel electrophoresis patterns of DNA treated with SmaI restriction enzyme were classified using Tenover's criteria and analyzed with the Fingerprinting II program to determine their genetic relatedness with the Colombian clone. RESULTS: All isolates had a genetic similarity of 78.5 percent or more with the Colombian clone. Thirteen electrophoretic subtypes derived of pattern A were identified, and five of them (A5, A6, A8, A13, A27) comprised 61.4 percent of the isolates. CONCLUSIONS: Clone Colombia5 ST289 is disseminated in Latin America. This is important because S. pneumoniae serotype 5 frequently causes invasive disease in the region and is associated with trimethoprim-sulfamethoxazole resistance.

OBJETIVO: Determinar la relación genética del clon Colombia5 ST289 con los aislamientos invasores de Streptococcus pneumoniae serotipo 5 provenientes de nueve países latinoamericanos. MÉTODOS: Se estudiaron 45 aislamientos invasores de Streptococcus pneumoniae serotipo 5 procedentes de niños menores de 5 años de Bolivia, Chile, Ecuador, Nicaragua, Panamá, Paraguay, Perú, República Dominicana y Venezuela. Los patrones en electroforesis en gel de campo pulsante del ADN tratado con la enzima de restricción SmaI se clasificaron mediante el criterio de Tenover y se analizaron con el programa Fingerprinting II para determinar su relación genética con el clon colombiano. RESULTADOS: Todos los aislamientos tuvieron una similitud genética de 78,5 por ciento o mayor con el clon colombiano. Se identificaron 13 subtipos electroforéticos derivados del patrón A y cinco de ellos (A5, A6, A8, A13 y A27) constituyeron 61,4 por ciento de los aislamientos. CONCLUSIONES: El clon Colombia5 ST289 está diseminado por América Latina. Esto es importante ya que S. pneumoniae serotipo 5 es causa frecuente de enfermedades invasoras en la Región y está asociado con la resistencia a trimetoprim-sulfametoxazol.