Genomic surveillance of invasive Streptococcus pneumoniae isolates in the period pre-PCV10 and post-PCV10 introduction in Brazil.

In 2010, Brazil introduced the 10-valent pneumococcal conjugate vaccine (PCV10) into the national children's immunization programme. This study describes the genetic characteristics of invasive Streptococcus pneumoniae isolates before and after PCV10 introduction. A subset of 466 [pre-PCV10 (2008-2009): n=232, post-PCV10 (2012-2013): n=234;<5 years old: n=310, ≥5 years old: n=156] pneumococcal isolates, collected through national laboratory surveillance, were whole-genome sequenced (WGS) to determine serotype, pilus locus, antimicrobial resistance and genetic lineages. Following PCV10 introduction, in the <5 years age group, non-vaccine serotypes (NVT) serotype 3 and serotype 19A were the most frequent, and serotypes 12F, 8 and 9 N in the ≥5 years old group. The study identified 65 Global Pneumococcal Sequence Clusters (GPSCs): 49 (88 %) were GPSCs previously described and 16 (12 %) were Brazilian clusters. In total, 36 GPSCs (55 %) were NVT lineages, 18 (28 %) vaccine serotypes (VT) and 11 (17 %) were both VT and NVT lineages. In both sampling periods, the most frequent lineage was GPSC6 (CC156, serotypes 14/9V). In the <5 years old group, a decrease in penicillin (P=0.0123) and cotrimoxazole (P<0.0001) resistance and an increase in tetracycline (P=0.019) were observed. Penicillin nonsusceptibility was predicted in 40 % of the isolates; 127 PBP combinations were identified (51 predicted MIC≥0.125 mg l-1); cotrimoxazole (folA and/or folP alterations), macrolide (mef and/or ermB) and tetracycline (tetM, tetO or tetS/M) resistance were predicted in 63, 13 and 21.6 % of pneumococci studied, respectively. The main lineages associated with multidrug resistance in the post-PCV10 period were composed of NVT, GPSC1 (CC320, serotype 19A), and GPSC47 (ST386, serotype 6C). The study provides a baseline for future comparisons and identified important NVT lineages in the post-PCV10 period in Brazil.

Long-term effect of 10-valent pneumococcal conjugate vaccine on nasopharyngeal carriage of Streptococcus pneumoniae in children in Brazil.

Brazil introduced the 10-valent pneumococcal vaccine (PCV10) to the routine national immunization program (NIP) in March 2010. In 2017, we investigated the effects of PCV10 on nasopharyngeal carriage of vaccine-types (VT) and non-vaccine-types (NVT) of Streptococcus pneumoniae (Spn) among children living in São Paulo city. We also compared the prevalence of VT and NVT with previous carriage surveys performed in 2010 (baseline) and 2013. METHOD: The carriage survey was conducted among 531 children, aged 12 months to <24 months, recruited from public Primary Health Units during the immunization campaign, using previous surveys methodology, except for qPCR, which was performed in the 2017 survey only. RESULTS: No statistical difference was found in the prevalence of Spn either by culture (59.7%) or by qPCR (61.2%). Spn carriage increased from 40.3% (baseline) to 59.7% (2017 survey) (p < 0.001). Colonization by VT isolates significantly decreased by 90.9% (19.8-1.8%) and 95.5% (19.8-0.9%) in the 2013 and 2017 surveys, respectively, compared to that at baseline. NVT isolates increased significantly by 128% (19.6-44.8%) and 185% (19.6-55.9%) in the respective post-PCV10 surveys, most led to high prevalence of serotypes 6C (27%), 15B (9.8%), 19A (9.2%), 15A (6.0%), and 16F (5.7%). In 2017, reduction in serotype 6A (4.2-0.6%, p < 0.001) and increase in serotype 19A (1.8-6.0%, p = 0.001) were found; serotype 3 isolate was not detected in the present survey. We identified the emergence of 19A isolates CC320, associated with high penicillin (MIC ≥ 2.0 mg/L) and cefotaxime (MIC ≥ 1.0 mg/L) values. CONCLUSION: After 7 years of PCV10 introduction in the NIP, colonization by VT among toddlers decreased substantially to a residual level, along with substantial serotype replacement by novel serotypes not present in any current conjugated pneumococcal vaccine and serotype 19A. The present findings can assist policy decisions in Brazil.

Long-term effect of 10-valent pneumococcal conjugate vaccine on nasopharyngeal carriage of Streptococcus pneumoniae in children in Brazil

Brazil introduced the 10-valent pneumococcal vaccine (PCV10) to the routine national immunization program (NIP) in March 2010. In 2017, we investigated the effects of PCV10 on nasopharyngeal carriage of vaccine-types (VT) and non-vaccine-types (NVT) of Streptococcus pneumoniae (Spn) among children living in São Paulo city. We also compared the prevalence of VT and NVT with previous carriage surveys performed in 2010 (baseline) and 2013. Method: The carriage survey was conducted among 531 children, aged 12 months to <24 months, recruited from public Primary Health Units during the immunization campaign, using previous surveys methodology, except for qPCR, which was performed in the 2017 survey only. Results: No statistical difference was found in the prevalence of Spn either by culture (59.7%) or by qPCR (61.2%). Spn carriage increased from 40.3% (baseline) to 59.7% (2017 survey) (p < 0.001). Colonization by VT isolates significantly decreased by 90.9% (19.8­1.8%) and 95.5% (19.8­0.9%) in the 2013 and 2017 surveys, respectively, compared to that at baseline. NVT isolates increased significantly by 128% (19.6­44.8%) and 185% (19.6­55.9%) in the respective post-PCV10 surveys, most led to high prevalence of serotypes 6C (27%), 15B (9.8%), 19A (9.2%), 15A (6.0%), and 16F (5.7%). In 2017, reduction in serotype 6A (4.2­0.6%, p < 0.001) and increase in serotype 19A (1.8­6.0%, p = 0.001) were found; serotype 3 isolate was not detected in the present survey. We identified the emergence of 19A isolates CC320, associated with high penicillin (MIC 2.0 mg/L) and cefotaxime (MIC 1.0 mg/L) values. Conclusion: After 7 years of PCV10 introduction in the NIP, colonization by VT among toddlers decreased substantially to a residual level, along with substantial serotype replacement by novel serotypes not present in any current conjugated pneumococcal vaccine and serotype 19A. The present findings can assist policy decisions in Brazil

Effect of 10-valent pneumococcal conjugate vaccine on nasopharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenzae among children in São Paulo, Brazil.

In March 2010, Brazil introduced the 10-valent pneumococcal conjugate vaccine (PCV10) in the routine infant immunization program using a 4-dose schedule and catch-up for children <23months. We investigated PCV10 effect on nasopharyngeal carriage with vaccine-type Streptococcus pneumoniae (Spn) and non-typeable Haemophilus influenzae (NTHi) among children in São Paulo city. Cross-sectional surveys were conducted in 2010 (baseline) and 2013 (post-PCV10). Healthy PCV-naïve children aged 12-23months were recruited from primary health centers during immunization campaigns. Nasopharyngeal swabs were collected and tested for Hi; for Spn, all baseline and a stratified random sample of 400 post-PCV10 swabs were tested. We compared vaccine-type Spn and NTHi carriage prevalence pre-/post-PCV10, and used logistic regression to estimate PCV10 effectiveness (1-adjusted odds ratio×100%). Overall 501 children were included in the baseline and 1167 in the post-PCV10 survey (including 400 tested for Spn). Spn was detected in 40.3% of children at baseline and 48.8% post-PCV10; PCV10 serotypes were found in 19.8% and 1.8% respectively, representing a decline of 90.9% (p<0.0001). Carriage of vaccine-related serotypes increased (10.8-21.0%, p<0.0001), driven primarily by a rise in serotype 6C (1.8-11.2%, p<0.0001); carriage of serotypes 6A and 19A did not significantly change. PCV10 effectiveness (4 doses) against vaccine-type carriage was 97.3% (95% confidence interval 88.7-99.3). NTHi prevalence increased from 26.0% (130/501) to 43.6% (509/1167, p<0.0001); PCV10 vaccination seemed significantly associated with NTHi carriage, even after adjusting for other known risk factors. Carriage with PCV10 serotypes among toddlers declined dramatically following PCV10 introduction in São Paulo, Brazil. No protection of PCV10 against NTHi was observed. Our findings contribute to a growing body of evidence of PCV10 impact on vaccine-type carriage and highlight the importance of PCV10 as a tool to reduce the burden of pneumococcal disease in Brazil and globally.

Caracterización fenotípica y molecular de neisseria meningitidis del serogrupo C asociada con un brote en Bahia, Brasil / Phenotypic and molecular characterization of serogroup C Neisseria meningitidis associated with an outbreak in Bahia, Brazil

Objetivo Caracterizar las cepas de Neisseria meningitidis (Nm) aisladas de cinco pacientes con Enfermedad Meningocócica (EM) asociada a un brote epidémico en Trancoso - BA, que ocurrió en octubre del 2009, luego de una fiesta en zona rural y en la que participaron 1000 jóvenes .Todos los casos fueron secundarios al caso primario a excepción de un paciente varón de 39 años. Materiales y métodos El Servicio de Vigilancia Epidemiológica del Estado de Bahia realizó la investigación epidemiológica y las cepas de Nm se caracterizaron en el Laboratorio Nacional de Referencia para Meningitis, Instituto Adolfo Lutz - São Paulo mediante métodos convencionales (sero - subtipificación y prueba de sensibilidad a los antimicrobianos) y métodos moleculares (electroforesis en gel de campo pulsado- PFGE y Multilocus Sequence Typing - MLST).Resultados La PFGE mostró dos perfiles de restricción estrechamente relacionados designados como PFGE tipos A y A1 con 92% de relación entre sí. Ambos tipos fueron clasificados como ST-3780 mediante MLST, y pertenecientes al complejo clonal ST-103. Todos los aislados mostraron el fenotipo C: 23: P1.5 y eran susceptibles a todos los antibióticos testados. Conclusiones Este es el primer brote de EM reportado asociado a cepas de Nm serogrupo C del complejo clonal ST-103 y relacionado con el consumo de drogas en Brasil (AU)

Objective To characterize meningococcal strains isolated from five cases of meningococcal disease (MD) associated with an outbreak in Trancoso - BA, occurred in October 2009. All cases, with the exception of a 39-year-old male, attended a dance party with approximately 1000 youngsters in a rural site. Materials and methods The epidemiological investigation was conducted by the Epidemiological Surveillance Service of Bahia State. Meningococcal strains were characterized at Adolfo Lutz Institute, the Brazilian National Reference Laboratory for Bacterial Meningitis by conventional techniques (serotype, serosubtype and antimicrobial susceptibility test) and by molecular methods (Pulsed-field gel electrophoresis - PFGE and Multilocus Sequence Typing - MLST).Results The PFGE showed 2 closely related restriction profiles, designated as PFGE types A and A1, having 92% relatedness to each other. MLST characterization showed both A and A1 clones were ST-3780, which belongs to the ST-103 complex. All isolates displayed the phenotype C:23:P1.5 and were susceptible to all antibiotics tested. Conclusions This is the first reported MD outbreak associated with serogroup C ST-103 complex in Brazil, as well as the party and illicit drug-use associated outbreak (AU)

Phenotypic and molecular characterization of serogroup C Neisseria meningitidis associated with an outbreak in Bahia, Brazil.

OBJECTIVE: To characterize meningococcal strains isolated from five cases of meningococcal disease (MD) associated with an outbreak in Trancoso - BA, occurred in October 2009. All cases, with the exception of a 39-year-old male, attended a dance party with approximately 1000 youngsters in a rural site. MATERIALS AND METHODS: The epidemiological investigation was conducted by the Epidemiological Surveillance Service of Bahia State. Meningococcal strains were characterized at Adolfo Lutz Institute, the Brazilian National Reference Laboratory for Bacterial Meningitis by conventional techniques (serotype, serosubtype and antimicrobial susceptibility test) and by molecular methods (Pulsed-field gel electrophoresis - PFGE and Multilocus Sequence Typing - MLST). RESULTS: The PFGE showed 2 closely related restriction profiles, designated as PFGE types A and A1, having 92% relatedness to each other. MLST characterization showed both A and A1 clones were ST-3780, which belongs to the ST-103 complex. All isolates displayed the phenotype C:23:P1.5 and were susceptible to all antibiotics tested. CONCLUSIONS: This is the first reported MD outbreak associated with serogroup C ST-103 complex in Brazil, as well as the party and illicit drug-use associated outbreak.

Changes in serotype distribution of Haemophilus influenzae meningitis isolates identified through laboratory-based surveillance following routine childhood vaccination against H. influenzae type b in Brazil.

Following routine childhood vaccination against Haemophilus influenzae type b (Hib) disease in Brazil in 1999, passive laboratory surveillance reported increasing numbers of non-b serotypes and nontypeable H. influenzae (NTHi) from meningitis cases. To characterize this increase, we analyzed data on 3910 H. influenzae isolated from cerebrospinal fluid or blood from meningitis cases that were sent to the national reference laboratory for serotyping from 1990 to 2008. Hib accounted for 98% of H. influenzae meningitis isolates received during 1990-1999 versus 59% during 2000-2008, while non-b serotypes increased from 1% to 19% and NTHi increased from 2% to 22% of H. influenzae isolates received during the two periods. Higher proportions of non-b serotypes and NTHi than Hib were isolated from blood rather than cerebrospinal fluid. Estimated incidence rates for H. influenzae meningitis for Sao Paulo state remained below 1 case per million population during 2000-2008, although annual incidence of NTHi meningitis (mean, 0.03 cases per 100,000 population) increased in several age groups. Changes in surveillance for H. influenzae following introduction of Hib conjugate vaccine likely contributed to increased numbers of non-b and nontypeable H. influenzae meningitis isolates received at the national reference laboratory.

[Nasopharyngeal colonization by Streptococcus pneumoniae in children with sickle cell disease receiving prophylactic penicillin]. / Colonização nasofaríngea pelo Streptococcus pneumoniae em crianças com doença falciforme usando penicilina profilática.

OBJECTIVES: To determine the prevalence of nasopharyngeal pneumococcus colonization in children with sickle cell disease undergoing penicillin prophylaxis, to identify risk factors for colonization and to serotype and determine antibiotic resistance in pneumococci obtained from those children. METHODS: Between April 9, 2002 and February 28, 2003, 188 nasopharyngeal swabs were obtained from 98 children with sickle cell disease in follow-up at the Hospital São Paulo-Universidade Federal de São Paulo. Pneumococci were isolated and identified by standard methods. The minimal inhibitory concentration for penicillin was determined by the E-test method. Isolates were serotyped with the use of type-specific antisera for 46 different serotypes (Neufeld-Quellung reaction). RESULTS: The age of children ranged from 4 months to 17 years (median and standard deviation 6.8-/+4.7 years). Thirteen of the 98 children had nasopharyngeal pneumococcus colonization (13.3% prevalence). There was a significantly greater risk of colonization among children less than 2 years old (p = 0.02). Twenty-one percent of isolates had intermediate penicillin resistance. There were no isolates highly resistant to penicillin. All isolates were susceptible to erythromycin, ceftriaxone, or vancomycin. The most frequently identified serotypes were 18C and 23F. CONCLUSIONS: Penicillin prophylaxis reduced pneumococcal nasopharyngeal colonization and did not increase the prevalence of penicillin-resistant pneumococci in children with sickle cell disease. Penicillin can be used not only for prophylaxis, but also in the acute management of febrile states with these children.

Colonização nasofaríngea pelo Streptococcus pneumoniae em crianças com doença falciforme usando penicilina profilática / Nasopharyngeal colonization by Streptococcus pneumoniae in children with sickle cell disease receiving prophylactic penicillin

OBJETIVOS: Determinar a prevalência de colonização nasofaríngea pelo pneumococo em crianças com doença falciforme, em uso de profilaxia com penicilina; identificar fatores de risco para colonização; sorotipar as cepas isoladas e avaliar a resistência antimicrobiana. METODOLOGIA: Foram colhidos 188 suabes de nasofaringe de 98 crianças com doença falciforme em acompanhamento no Hospital São Paulo, da Universidade Federal de São Paulo, no período de 09 de abril de 2002 a 28 de fevereiro de 2003. O isolamento e a identificação dos pneumococos seguiram procedimentos padronizados. A concentração inibitória mínima para penicilina foi determinada pelo método do E-test. A sorotipagem foi realizada pela reação de Neufeld-Quellung com anti-soros para 46 sorotipos. RESULTADOS: A idade variou de 4 meses a 17 anos (média e desvio padrão de 6,8±4,7 anos). Das 98 crianças do estudo, 13 apresentaram colonização pelo pneumococo (prevalência de 13,3 por cento). O maior risco de colonização ocorreu em menores de 2 anos de idade (p = 0,02). A prevalência de cepas com resistência intermediária à penicilina foi de 21,4 por cento, não sendo evidenciada resistência plena. Também não houve cepas resistentes à eritromicina, ceftriaxona e vancomicina. Os sorotipos isolados mais freqüentes foram o 18C e o 23F. CONCLUSÕES: O uso profilático de penicilina diminuiu a colonização nasofaríngea pelo pneumococo e não determinou aumento da resistência a esse antimicrobiano nas crianças com doença falciforme. A penicilina ainda pode ser usada na profilaxia e no tratamento dos episódios febris dessas crianças.

Evaluation of methodology for serotyping invasive and nasopharyngeal isolates of Haemophilus influenzae in the ongoing surveillance in Brazil.

To assess the magnitude of discrepant results obtained by routine Haemophilus influenzae serotyping, 258 isolates, collected by the epidemiological surveillance system in Brazil from individuals with invasive diseases or carriage, were evaluated by two slide agglutination (SlAg) methods: SlAg method 1, by which strains were initially screened with a serotype b-specific antiserum, and SlAg method 2, by which strains were tested against all serotype-specific antisera in parallel. Investigators comparing results of the two SlAg methods with those obtained by capsule type-specific PCR were blinded to the method used. The serotype prevalence rates found by the three methods were significantly different, involving discrepancies mainly between serotype b and noncapsulated (NC) isolates. For invasive isolates (n = 131), the overall agreement rate between SlAg method 1 or 2 and PCR was 68.0 or 88.3%, respectively, whereas for colonizing isolates (n = 127) the corresponding rate was 46.5 or 94.2%, respectively. SlAg method 2 improved the ascertainment of serotypes over that obtained with SlAg method 1, demonstrating good correlation with PCR. Use of the polyvalent antiserum as a screening reagent for SlAg for invasive and colonizing isolates showed poor discriminatory power, with a sensitivity of 65.8% and a specificity of 91.7%. We stress the importance of using a well-standardized SlAg methodology and suggest that reference laboratories should utilize PCR routinely to confirm SlAg results and to check all nonspecific SlAg reactions and apparent NC isolates by SlAg in order to provide reliable data on the prevalence of H. influenzae serotypes in the H. influenzae type b vaccine era.

Streptococcus pneumoniae and Haemophilus influenzae as etiological agents of conjunctivitis outbreaks in the region of Riveiräo Preto, SP, Brazil

No estudo de surtos de conjuntivite ocorridos no periodo de setembro de 1994 a setembro de 1996, na regiäo de Ribeirao Preto, foram semeadas no Instituto Adolfo Lutz Laboratorio I, Ribeiräo Preto, exsudatos conjuntivais de 92 pacientes, sendo que a maioria dos casos estava na faixa etaria de 2-7 anos. Os agentes etiologicos mais frequentemente isolados foram dos casos analisados foram: Streptococcus pneumoniae e Haemophilus influenzae em 40,22 por cento e 21,74 por cento respectivamente. 51,35 por cento das cepas de S. pneumoniae isoladas foram näo tipaveis. As cepas de S. pneumoniae oxacilina resistente foram submetidas ao teste de concentracao inibitoria minima (CIM), sendo que tres apresentaram resistencia intermediaria e apenas uma foi altamente resistente a penicilina

Padronizaçäo de um modelo de peritonite em ratos / Standardization of a peritonitis model in rats

Para o estudo experimental das conseqüências metabólicas da infecçäo sistêmica de origem peritoneal os autores padronizaron um modelo em ratos. Prepararam-se cápsulas gelatinosas contendo quantidades conhecidas e proggressivas de 10**3 a 10**9 de E. coli em meio a bário e fezes estéreis de ratos. As cápsulas foram implantadas através de pequena laparotomia mediana na cavidade peritoneal de 54 ratos divididos em 6 grupos. Cada grupo recebeu uma quantidade diferente de E. coli. As hemoculturas realizadas por sangria do animal, após 6 horas de inoculaçäo da cápsula foram sempre positivas. A mortalidade dos animais até 48 horas pós implante da cápsula, guardou relaçäo com o número de E. coli inoculada, chegando a 100// com 8,8 x 10**7. Com 3,4 x 10**6 a 6,0 x 10**6 as hemoculturas indicaram bacteremia inicial que decaiu progressivamente com a recuperaçäo clínica do animal. Esses resultados indicaram que o implante intraperitoneal de uma cápsula com um número reconhecido de E. coli é um modelo consistente, homogênio e reprodutível para bacteremia experimental e septicemia