Distribuição de sorotipos, perfil de suscetibilidade antimicrobiana e caracterização molecular de cepas de streptococcus pneumoniae isoladas de doença pneumocócica invasiva nos períodos pré e pós a introdução da vacina pneumocócica 10-valente no Brasil / Distribution of serotypes, antimicrobial susceptibility profile and molecular characterisation of strains of Streptococcus pneumoniae isolated from invasive pneumococcal disease in the periods before and after the introduction of the 10-valent pneumococcal vaccine in Brazil

A doença pneumocócica invasiva (DPI) é uma das principais causas de morbidade e letalidade afetando especialmente crianças e idosos, resultando em um problema de saúde pública. O Brasil introduziu a vacina pneumocócica conjugada 10-valente (PCV10) no programa nacional de imunização infantil em 2010. Os estudos de efetividade e de impacto da PCV10 no Brasil foram realizados 3 a 5 anos após a introdução da vacina e mostraram redução nos casos de DPI causada pelos sorotipos vacinais e aumento de sorotipos não incluídos na vacina. Portanto, estudos realizados em um período de tempo mais longo após vacinação são fundamentais para se observar a sustentabilidade do aumento dos sorotipos não vacinais ao longo dos anos. O objetivo deste estudo foi investigar as características de S.pneumoniae (distribuição de sorotipos, perfil de suscetibilidade aos antimicrobianos e identificação das linhagens genéticas dos sorotipos prevalentes) nos períodos pré-PCV10 (2005-2009), pós-PCV10-imediato (2010-2013) e pós-PCV10-tardio (2014-2017), sendo o período pósPCV10 composto de 7 anos de avaliação. Isolados de DPI foram obtidos através da vigilância laboratorial nacional para S. pneumoniae. Os isolados foram encaminhados ao Instituto Adolfo Lutz pelos LACENs e por outras instituições públicas e privadas. Os isolados foram sorotipados por Quellung, o perfil de suscetibilidade antimicrobiana foi determinado pelos testes de disco-difusão e concentração inibitória mínima por microdiluição e a caracterização molecular foi realizada por MLST. A %change foi utilizada para calcular as diferenças na prevalência dos sorotipos e da resistência antimicrobiana por período de estudo. As sequências-tipo foram determinadas na página da web MLST e os complexos clonais pelo programa eBURST. Um total de 11.136 isolados invasivos foi estudado fenotipicamente. Uma amostragem de 688 isolados foi selecionada para a identificação das linhagens genéticas. No período pós-PCV10-tardio foi observada uma redução de 69,6% de DPI pelos sorotipos vacinais e, em paralelo, um aumento de 105,8% dos sorotipos não incluídos na PCV10. O aumento dos sorotipos não-PCV10 foi relacionado principalmente aos sorotipos 3, 6C, 8, 12F e 19A. Detectamos uma elevação de 304,6% na resistência à eritromicina no período pósPCV10-tardio. O estudo molecular identificou 33 CC e 182 STs. No período pós-PCV10, clones internacionalmente disseminados foram identificados ST180 (Clone Holanda3 -31), ST53-12574 (Clone Holanda8 - 33) e ST218 (Clone Dinamarca12F-34)], e duas principais STs foram relacionadas à resistência aos antimicrobianos, a citar a ST320/19A, presente desde o pré-PCV10 e a ST386/6C detectada no pós-PCV10. O monitoramento das características de S. pneumoniae em um período de tempo longo após a introdução da PCV10 confirmou a proteção da vacina contra a DPI pelos sorotipos vacinais e detectou a alta prevalência de sorotipos não incluídos na PCV10. O estudo molecular identificou uma disseminação de clones internacionais no Brasil

Invasive pneumococcal disease (IPD) is one of the leading causes of morbidity and lethality affecting especially children and the elderly, resulting in a public health problem. Brazil introduced the 10-valent pneumococcal conjugate vaccine (PCV10) in the national program of childhood immunization in 2010. The effectiveness and impact studies of PCV10 in Brazil were carried out 3 to 5 years after the introduction of the vaccine and showed a reduction in cases of IPD caused by vaccine serotypes and increase in non-vaccine serotypes. Therefore, studies conducted over a longer period of time after vaccination is essential to observe the sustainability of the increase in non-vaccine serotypes over the years. The aim of this study was to investigate the characteristics of S. pneumoniae (distribution of serotypes, antimicrobial susceptibility profile and genetic lineages identification of prevalent serotypes) in the pre-PCV10 (2005-2009), immediate-postPCV10 (2010-2013) and late-post-PCV10 (2014-2017) periods, the postPCV10 period being composed of 7 years of evaluation. Isolated of DPI were obtained through national laboratory surveillance for S. pneumoniae. The isolates were sent to the Institute Adolfo Lutz by LACENs and other public and private institutions. The isolates were serotype by Quellung, the antimicrobial susceptibility profile was determined by disc-diffusion and minimum inhibitory concentration microdilution assays and the molecular characterization were performed by MLST. %change was used to calculate differences in the prevalence of serotypes and antimicrobial resistance per study period. The sequences-type were determined at MLST website and clonal complexes were defined by the program eBURST. A total of 11,136 invasive isolates were phenotipically studied. A sample of 688 isolates was selected for the identification of the genetic lineages. In the latepost-PCV10 period, a 69.6% reduction in IPD was observed by vaccine serotypes and, in parallel, a 105.8% increase in non-PCV10 serotypes. The increase in non-PCV10 serotypes was mainly related to serotypes 3, 6C, 8, 12F and 19A. We detected a 304.6% increase in resistance to erythromycin in the late-post-PCV10 period. The molecular study identified 33 CC and 182 STs. In the post-PCV10 period, internationally disseminated clones were identified [ST180 (Clone Netherlands3-31), ST53-12574 (Clone Netherlands8-33) and ST218 (Clone Denmark12F34)], and two major STs were related to antimicrobial resistance, to be cited the ST320/19A, present since the pre-PCV10 and the ST386/6C detected post-PCV10. Monitoring the characteristics of S. pneumoniae a long-term period after the introduction of PCV10 confirmed the protection of the vaccine against IPD by the vaccine serotypes and detected the high prevalence of non-PCV10 serotypes. The molecular study identified the great spread of international clones in Brazil

Clinical and microbiological implications of invasive pneumococcal disease in hospitalized patients (1998-2013)

Abstract Introduction Infections caused by Streptococcus pneumoniae (pneumococcus) still represent a challenge for health systems around the world. Objective The objective of this study was to assess microbiological and clinical aspects in hospitalized patients with invasive pneumococcus disease between 1998 and 2013. Materials and methods This was a retrospective study that analyzed the results of pneumococcus identification, serotyping, and susceptibility testing found in the Adolfo Lutz Institute databank. Personal variables, medical history and clinical outcome of patients admitted with invasive pneumococcal disease were analyzed. These were obtained from records of a public teaching hospital – Hospital das Clínicas Faculdade de Medicina Ribeirão Preto. Results The sample comprised 332 patients. Patient age ranged from less than one month to 89 years old (mean 20.3 years) and the sample was predominately male. Pneumonia (67.8%) was the most common disease, accounting for 18.2% of deaths. Serotypes 14, 1, 3, 9V, 6B, 6A, 23F, 19A, 18C, 19F, 12F, and 4 were the most common (75.3%). Most patients, or 67.5%, were cured without any complication (success), 6.9% had some type of sequela (failure), and 25.6% died (failure). In the case of deaths due to meningitis, strains of fully penicillin resistant pneumococcus were isolated. Furthermore, 68.2% of patients who died presented some type of comorbidity. The 60 and older age group presented the most significant association (Odds Ratio = 4.2), with outcome failure regardless of the presence of comorbidity. Serotype 18C was the most significant risk factor both in raw analysis (Odds Ratio = 3.8) and when adjusted for comorbidity (Odds Ratio = 5.0) or age (Odds Ratio = 5.4). The same occurred with serotype 12F (respectively, Odds Ratio = 5.1, Odds Ratio = 5.0, and Odds Ratio = 4.7) Conclusion The present findings highlight the importance of IPD among young adults and older adults. In the era of conjugate vaccines, monitoring serotypes in different age groups is essential to assess the impact and adequacy of immunization.

Evaluation of Haemophilus influenzae type b carrier status among children 10 years after the introduction of Hib vaccine in Brazil

The aim of the present study was to assess the prevalence of Haemophilus influenzaetype b (Hib) nasopharyngeal (NP) colonisation among healthy children where Hib vaccination using a 3p+0 dosing schedule has been routinely administered for 10 years with sustained coverage (> 90%). NP swabs were collected from 2,558 children who had received the Hib vaccine, of whom 1,379 were 12-< 24 months (m) old and 1,179 were 48-< 60 m old. Hi strains were identified by molecular methods. Hi carriage prevalence was 45.1% (1,153/2,558) and the prevalence in the 12-< 24 m and 48-< 60 m age groups were 37.5% (517/1,379) and 53.9% (636/1,179), respectively. Hib was identified in 0.6% (16/2,558) of all children in the study, being 0.8% (11/1,379) and 0.4% (5/1,179) among the 12-< 24 m and 48-< 60 m age groups, respectively. The nonencapsulate Hi colonisation was 43% (n = 1,099) and was significantly more frequent at 48-< 60 m of age (51.6%, n = 608) compared with that at 12-< 24 m of age (35.6%, n = 491). The overall resistance rates to ampicillin and chloramphenicol were 16.5% and 3.7%, respectively; the co-resistance was detected in 2.6%. Our findings showed that the Hib carrier rate in healthy children under five years was very low after 10 years of the introduction of the Hib vaccine.

Prevalence of pneumococcal serotypes and resistance to antimicrobial agents in patients with meningitis: ten-year analysis

OBJECTIVES: To determine the prevalence of pneumococcal serotypes and antimicrobial susceptibility in patients with meningitis, and to evaluate the implications for vaccine coverage. METHODS: Pneumococcal strains obtained from normally sterile fluids from patients admitted with meningitis were isolated at the Hospital de Clínicas of the Universidade Federal de Uberlândia, Minas Gerais State, and sent to the Instituto Adolfo Lutz, city of São Paulo, São Paulo State, for further identification, serotyping, and antimicrobial susceptibility determination. RESULTS: From April 1999 to April 2009, 338 pneumococcal strains were isolated, and 72 obtained from patients with meningitis, were analyzed. Patients' ages varied from one month to 82.2 years (mean of 18.4 ± 22.9 years; median of 5.2 years) and 46 (63.9 percent) patients were male. Strains were isolated from cerebrospinal fluid [66 occasions (91.7 percent)] and blood [6 occasions (8.3 percent)]. The most commonly identified serotypes were 14, 19F, 3, 7F, 6A, 6B, 10A, 18C, 23F, 5, and 34. Of the 20 [27.8 percent] oxacillin-resistant strains, 17 [23.6 percent] were resistant to penicillin and nine [12.5 percent] to ceftriaxone, both resistance patterns being more common in children aged two years or less and during the 2005-2009 period. CONCLUSIONS: Resistance to penicillin and ceftriaxone was detected in 23.6 percent and 12.5 percent of the strains, respectively, and predominated in children aged two years or less and during the 2005-2009 period. There were 24 different serotypes of pneumococcus and 79.8 percent of the serotypes were represented in the 7-valent conjugated vaccine [PVC7].

Prevalência de sorotipos e resistência antimicrobiana de cepas invasivas do pneumococo em crianças: análise de 9 anos / Prevalence of serotypes and antimicrobial resistance of invasive strains of pneumococcus in children: analysis of 9 years

OBJETIVO: Avaliar o perfil de sorotipos e a sensibilidade aos antimicrobianos de cepas de pneumococo obtidas de crianças e as implicações na formulação de vacinas pneumocócicas. MÉTODOS: Cepas de pneumococo isoladas no Hospital de Clínicas da Universidade Federal de Uberlândia, Uberlândia (MG), a partir de pacientes com doença invasiva, foram enviadas ao Instituto Adolfo Lutz, São Paulo (SP), para confirmação da identificação, sorotipagem e determinação da sensibilidade aos antimicrobianos. RESULTADOS: De abril de 1999 a dezembro de 2008, foram avaliadas 142 cepas de pneumococo obtidas de crianças de até 5 anos de idade. Setenta e cinco (52,8 por cento) eram de pacientes do sexo masculino, e a idade variou de 1 a 60 meses (média de 19±15,4 meses e mediana de 15 meses). Os diagnósticos clínicos mais comuns foram pneumonia [92 casos (64,8 por cento)] e meningite [33 casos (23,2 por cento)], e as principais fontes de recuperação foram sangue [61 amostras (43 por cento)], líquido pleural [52 (36,6 por cento)] e liquor [28 (19,7 por cento)]. Os sorotipos mais comuns foram o 14, 5, 6B, 1, 6A, 18C, 19A, 3, 9V, 19F, 23F, 9N e 10A. Foram detectadas 14 (9,9 por cento) cepas penicilina-resistentes, restritas aos sorotipos 14, 6B, 19F, 19A e 23F e predominantes no período de 2004 a 2008 (p = 0,000). Foi detectada sensibilidade diminuída ao cotrimoxazol (79,5 por cento), à eritromicina e à clindamicina (11,3 por cento cada) e à ceftriaxona (5,6 por cento). CONCLUSÕES: A resistência à penicilina foi detectada em 9,9 por cento das cepas e predominou no período de 2004 a 2008. Foram identificados 20 diferentes sorotipos de pneumococo, e a cifra de cobertura pela vacina 7-valente atualmente disponível (PN CRM7) é de 71,9 por cento.

OBJECTIVE: To determine the prevalence of serotypes and antimicrobial susceptibility of strains of pneumococcus in children and to evaluate the implications for vaccine formulation. METHODS: Strains of pneumococcus obtained from children admitted with invasive diseases were isolated at Hospital de Clínicas of Universidade Federal de Uberlândia, Uberlândia, Brazil, and sent to Instituto Adolfo Lutz, São Paulo, Brazil, for further identification, serotyping, and determination of antimicrobial susceptibility. RESULTS: From April 1999 to December 2008, 142 strains of pneumococcus, obtained from children under 5 years of age, were analyzed. Seventy-five (52.8 percent) patients were male, and the age ranged from 1 to 60 months (mean age = 19±15.4 months; median = 15 months). The most common diagnoses were pneumonia [92 cases (64.8 percent)] and meningitis [33 cases (23.2 percent)]. The strains were mostly isolated from blood [61 samples (43 percent)], pleural fluid [52 samples (36.6 percent)], and cerebrospinal fluid [28 samples (19.7 percent)]. The most common serotypes were 14, 5, 6B, 1, 6A, 18C, 19A, 3, 9V, 19F, 23F, 9N, and 10A. There were 14 [9.9 percent] penicillin-resistant strains, which was detected only in the following serotypes: 14, 6B, 19F, 19A, and 23F, being predominant from 2004 to 2008 (p = 0.000). There was reduced susceptibility to co-trimoxazole (79.5 percent), erythromycin and clindamycin (11.3 percent each), and ceftriaxone (5.6 percent). CONCLUSIONS: Penicillin resistance was detected in 9.9 percent of the strains, being predominant from 2004 to 2008. Twenty different pneumococcal serotypes were identified, and 71.9 percent of the serotypes were represented in the 7-valent conjugate vaccine (PN CRM7) currently available.

Novos pontos de corte de sensibilidade nas taxas de resistência antimicrobiana de cepas invasivas de pneumococo / New susceptibility breakpoints in antimicrobial resistance rates of invasive pneumococcal strains

OBJETIVO: Avaliar impacto dos novos pontos de corte de sensibilidade à penicilina nas taxas de resistência de cepas de pneumococo obtidas de crianças com pneumonia. MÉTODOS: Cepas de pneumococo isoladas no laboratório de análises clínicas do Hospital de Clínicas de Uberlândia, Uberlândia (MG), a partir de amostras de pacientes internados foram enviadas ao Instituto Adolfo Lutz, Sao Paulo (SP), para confirmação da identificação, sorotipagem e determinação da sensibilidade aos antimicrobianos. RESULTADOS: De abril de 1999 a dezembro de 2008 foram enviadas ao Instituto Adolfo Lutz 330 cepas de pneumococo, sendo 195 (59 por cento) provenientes de pacientes com diagnóstico de pneumonia. Destas, foram analisadas 100 cepas de pacientes com idade ≤ 12 anos; a idade dos pacientes variou de 1 a 12,6 anos, com média de 2,4 e mediana de 1,7 anos; 47 pacientes eram do sexo masculino; as fontes de recuperação foram sangue (42 por cento) e líquido pleural (58 por cento). Foram detectadas 35 cepas oxacilina-resistentes: segundo os critérios do Clinical and Laboratory Standards Institute (CLSI) de 2007 [concentração inibitória mínima (CIM) ≤ 0,06 µg/mL para sensibilidade (S), 0,12 a 1 µg/mL para resistência intermediária (RI) e ≥ 2 µg/mL para resistência plena (RP)], 22 cepas apresentaram RI e 11, RP para penicilina. De acordo com os critérios atuais do CLSI de 2008 (≤ 2 µg/mL para S, 4 µg/mL para RI e ≥ 8 µg/mL para RP) apenas uma cepa confirmou RI à penicilina. Detectou-se resistência a cotrimoxazol (80 por cento), tetraciclina (21 por cento), eritromicina (13 por cento), clindamicina (13 por cento) e ceftriaxona (uma cepa, simultaneamente resistente a penicilina). CONCLUSÕES: Com a aplicação dos novos pontos de corte para sensibilidade in vitro, as taxas de resistência a penicilina caíram 97 por cento, de 33 para 1 por cento.

OBJECTIVE: To evaluate the impact of new penicillin susceptibility breakpoints on resistance rates of pneumococcal strains collected from children with pneumonia. METHODS: Pneumococcal strains collected from patients admitted with pneumonia were isolated at the clinical analysis lab of Hospital de Clínicas de Uberlândia, Uberlândia, Brazil, and sent to Instituto Adolfo Lutz, São Paulo, Brazil, for further identification, serotyping and determination of antimicrobial susceptibility. RESULTS: From April 1999 to December 2008, 330 strains of pneumococcus were sent to Instituto Adolfo Lutz; of these, 195 (59 percent) were collected from patients with pneumonia. One hundred strains collected from patients ≤ 12 years old were analyzed. The patients' age ranged from 1 to 12.6 years old (with mean age of 2.4 and median of 1.7 years). Forty-seven patients were male. The strains were isolated from blood (42 percent) and pleural fluid (58 percent). There were 35 oxacillin-resistant strains: according to the criteria defined by the Clinical and Laboratory Standards Institute (CLSI) in 2007 [minimum inhibitory concentration (MIC) ≤ 0.06 µg/mL for susceptibility (S), 0.12 to 1 µg/mL for intermediate resistance (IR), and ≥ 2 µg/mL for full resistance (FR)], 22 strains had IR and 11 strains had FR. According to the current breakpoints defined by the CLSI in 2008 (≤ 2 µg/mL for S, 4 µg/mL for IR and ≥ 8 µg/mL for FR), only one strain had IR to penicillin. There was resistance to co-trimoxazole (80 percent), tetracycline (21 percent), erythromycin (13 percent), clindamycin (13 percent), and ceftriaxone (one strain simultaneously resistant to penicillin). CONCLUSIONS: When the new breakpoints for in vitro susceptibility were applied, penicillin resistance rates dropped 97 percent, from 33 to 1 percent.