RESUMO
The incorporation of Haemophilus influenzae type b (Hib) vaccine into the Argentine National Immunization Program in 1998 resulted in a dramatic decrease in the incidence of invasive disease due to this serotype. We assessed 1405 H. influenzae (Hi) isolates causing invasive infections referred to the National Reference Laboratory between 2011 and 2019. Non-encapsulated Hi were the most common strains (44.5%), followed by types b (41.1%) and a (10.0%). Significant increase in the proportion of type b was observed, from 31.2% in 2011, to 50% in 2015, correlating with the peak incidence rate, later decreasing to 33.6% by 2019. We compared the genetic relationship between clones circulating during the period of increased Hib incidence (2011-2015) and those of the prevaccination-transition period (1997-1998). Four pulsotypes predominated in both periods, G, M, P and K, G being the most common. Multi-locus sequence typing revealed that the 4 pulsotypes belonged to ST6, or one of its simple or double locus variants. Isolates from fully vaccinated individuals did not differ from those of the rest of the population studied. After ruling out aspects associated with emergence of specific clones, we concluded that factors such as low booster coverage rates, delayed vaccination schedules and use of different vaccines may have contributed to the reemergence of Hib infections.
Assuntos
Infecções por Haemophilus , Vacinas Anti-Haemophilus , Haemophilus influenzae tipo b , Humanos , Lactente , Haemophilus influenzae tipo b/genética , Tipagem de Sequências Multilocus , Argentina/epidemiologia , Infecções por Haemophilus/epidemiologia , Infecções por Haemophilus/prevenção & controle , Haemophilus influenzae/genética , IncidênciaRESUMO
Since the introduction of Haemophilus influenzae (Hi) serotype b (Hib) vaccination, reports of increasing incidence rates of non-Hib serotypes have emerged. A systematic review was performed to investigate whether the Hi serotype f (Hif) incidence rate has increased globally and to describe its associated disease burden. In the post-Hib vaccine era, evidence shows that the incidence rate of Hif infection is increasing worldwide. In total 94 studies including 2â701 patients reported Hif infections. The estimated pooled incidence rate of Hif infection was 0.15/100â¯000 population per year (range: 0.05-0.40/100â000), with a median case fatality ratio of 14.3â%. Invasive infections most frequently presented as pneumonia (45â%), septicaemia (34â%) and meningitis (20â%). Of 191 Hif isolates, 87â% were ampicillin-susceptible. Multi-locus sequence typing revealed that Hif were relatively clonal, with the majority belonging to clonal complex 124. Hif causes invasive infections of significant variance in both severity and presentation. Globally, the Hif population shows little genetic variability and currently appears to possess low resistance to antimicrobials.
Assuntos
Infecções por Haemophilus , Vacinas Anti-Haemophilus , Haemophilus influenzae tipo b , Humanos , Lactente , Haemophilus influenzae tipo b/genética , Infecções por Haemophilus/epidemiologia , Infecções por Haemophilus/prevenção & controle , Tipagem de Sequências Multilocus , Haemophilus influenzae , Ampicilina , VacinaçãoRESUMO
BACKGROUND: The epidemiologic characteristics of invasive Haemophilus influenzae type b disease (HIBD) have markedly changed since the introduction of the Haemophilus influenzae type b (Hib) conjugate vaccine worldwide. The immunization schedule against Haemophilus influenzae type b differs in Europe. METHODS: This is a retrospective observational study which evaluates all the data included in the molecular surveillance register for invasive infectious diseases at the Laboratory of Molecular Diagnosis at Meyer Children's University Hospital from December 2008 to December 2018 with a diagnosis of invasive HIBD in children <5 years of age. RESULTS: We identified 4 cases of HIBD: all the cases presented signs or symptoms of invasive infection and the H. influenzae type b was identified in cerebrospinal fluid, or blood or bronchoalveolar lavage by molecular test. The crude incidence for Hib invasive disease in Tuscany is 0.26/100,000 p-y in children younger than 5 years, significantly different from the incidence rate before the introduction of the Hib vaccination. Vaccination effectiveness can be estimated at 97.9% and the impact of hexavalent (2p+1) vaccine at 99.6%. CONCLUSIONS: This work confirms the high impact of the hexavalent vaccine 2p+1 schedule for HIBD in children <5 years, emphasizing the role of molecular test for HIBD diagnosis and surveillance.
Assuntos
Monitoramento Epidemiológico , Infecções por Haemophilus/epidemiologia , Vacinas Anti-Haemophilus/imunologia , Haemophilus influenzae tipo b/genética , Líquido da Lavagem Broncoalveolar/microbiologia , Pré-Escolar , Infecções por Haemophilus/sangue , Infecções por Haemophilus/líquido cefalorraquidiano , Haemophilus influenzae tipo b/isolamento & purificação , Haemophilus influenzae tipo b/patogenicidade , Humanos , Esquemas de Imunização , Incidência , Lactente , Recém-Nascido , Itália/epidemiologia , Estudos Retrospectivos , Vacinas Combinadas/imunologiaRESUMO
Haemophilus influenzae remains a common cause of illness in children worldwide. H. influenzae type b is the leading cause of bacterial meningitis in children before introduction of vaccination and is a common cause of pneumonia, epiglottis and septic arthritis. Since the implementation of the Hib conjugate vaccine, the non-typeable H. influenzae has rapidly decreased in respiratory and invasive infections in children and adults. However, the rate of antibiotic resistance of H. influenzae varies with region and period and is usually on the rise. In this review, typing of H. influenzae, virulence factors and resistance will be dissertated.
Assuntos
Evolução Molecular , Infecções por Haemophilus/epidemiologia , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/classificação , Haemophilus influenzae/genética , Antibacterianos/farmacologia , Biofilmes , Farmacorresistência Bacteriana , Infecções por Haemophilus/prevenção & controle , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/imunologia , Haemophilus influenzae tipo b/genética , Haemophilus influenzae tipo b/imunologia , Humanos , Epidemiologia Molecular , Polissacarídeos Bacterianos/imunologia , Sorotipagem , Virulência/genética , Fatores de Virulência/genética , Resistência beta-LactâmicaRESUMO
BACKGROUND: Since the 1990s, the epidemiology of bacterial meningitis worldwide has changed thanks to vaccination. In Tunisia, the main causative pathogens were Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae serotype b (Hib). Only Hib vaccination was available during our study period. OBJECTIVES: We performed a laboratory case report based-study of suspected bacterial meningitis in Northern Tunisia from January 2014 to June 2017. METHODS: CSF samples obtained from children beyond neonatal age with suspicion of meningitis were tested by two real time PCRs, targeting pneumococcus, meningococcus and Hib, and conventional methods. RESULTS: Using real-time PCR, 63 were positive including ten supplementary cases compared to conventional methods. A general decrease of bacterial meningitis cases was demonstrated comparing to previous data. Pneumococcus was predominant (69.84%) followed by meningococcus (28.57%) and Hib (1.59%). The main serotypes were 14, 19F, 6B and 23F for pneumococcus and serogroup B for meningococcus. Most cases occurred during cold season and children under one year were the most affected by bacterial meningitis. CONCLUSION: Our study suggests the predominance of pneumococcal cases. It may provide valuable data on meningitis epidemiology before the introduction of pneumococcal vaccine, which may be useful for future evaluation.
Assuntos
Líquido Cefalorraquidiano/microbiologia , Haemophilus influenzae tipo b/genética , Meningites Bacterianas/epidemiologia , Neisseria meningitidis/genética , Streptococcus pneumoniae/genética , Criança , Pré-Escolar , DNA Bacteriano , Estudos Epidemiológicos , Feminino , Haemophilus influenzae tipo b/isolamento & purificação , Humanos , Lactente , Recém-Nascido , Masculino , Meningites Bacterianas/líquido cefalorraquidiano , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/microbiologia , Neisseria meningitidis/isolamento & purificação , Vigilância da População , Reação em Cadeia da Polimerase em Tempo Real , Estações do Ano , Streptococcus pneumoniae/isolamento & purificação , Tunísia/epidemiologiaAssuntos
Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/administração & dosagem , Infecções por Haemophilus/prevenção & controle , Vacinas Anti-Haemophilus/administração & dosagem , Haemophilus influenzae tipo b/imunologia , Polissacarídeos Bacterianos/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Feminino , Infecções por Haemophilus/imunologia , Infecções por Haemophilus/microbiologia , Vacinas Anti-Haemophilus/genética , Vacinas Anti-Haemophilus/imunologia , Haemophilus influenzae tipo b/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Polissacarídeos Bacterianos/imunologiaRESUMO
BACKGROUND: Multiplex real-time polymerase chain reaction assays have improved diagnostic sensitivity for a wide range of pathogens. However, co-detection of multiple agents and bacterial colonization make it difficult to distinguish between asymptomatic infection or illness aetiology. We assessed whether semi-quantitative microbial load data can differentiate between symptomatic and asymptomatic states for common respiratory pathogens. METHODS: We obtained throat and nasal swab samples from military trainees at two Thai Army barracks. Specimens were collected at the start and end of 10-week training periods (non-acute samples), and from individuals who developed upper respiratory tract infection during training (acute samples). We analysed the samples using a commercial multiplex respiratory panel comprising 33 bacterial, viral and fungal targets. We used random effects tobit models to compare cycle threshold (Ct) value distributions from non-acute and acute samples. RESULTS: We analysed 341 non-acute and 145 acute swab samples from 274 participants. Haemophilus influenzae type B was the most commonly detected microbe (77.4% of non-acute and 64.8% of acute samples). In acute samples, nine specific microbe pairs were detected more frequently than expected by chance. Regression models indicated significantly lower microbial load in non-acute relative to acute samples for H. influenzae non-type B, Streptococcus pneumoniae and rhinovirus, although it was not possible to identify a Ct-value threshold indicating causal etiology for any of these organisms. CONCLUSIONS: Semi-quantitative measures of microbial concentration did not reliably differentiate between illness and asymptomatic colonization, suggesting that clinical symptoms may not always be directly related to microbial load for common respiratory infections.
Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Infecções Respiratórias/diagnóstico , Doença Aguda , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Feminino , Haemophilus influenzae tipo b/genética , Haemophilus influenzae tipo b/isolamento & purificação , Humanos , Masculino , Militares , Cavidade Nasal/microbiologia , Faringe/microbiologia , Estudos Prospectivos , RNA Viral/genética , RNA Viral/metabolismo , Infecções Respiratórias/microbiologia , Infecções Respiratórias/virologia , Rhinovirus/genética , Rhinovirus/isolamento & purificação , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação , TailândiaRESUMO
Military recruits are at high risk of respiratory infections. However, limited data exist on military populations in tropical settings, where the epidemiology of respiratory infections differs substantially from temperate settings. We enrolled recruits undertaking a 10-week military training at two Royal Thai Army barracks between May 2014 and July 2015. We used a multiplex respiratory panel to analyze nose and throat swabs collected at the start and end of the training period, and from participants experiencing respiratory symptoms during follow-up. Paired sera were tested for influenza seroconversion using a hemagglutinin inhibition assay. Overall rates of upper respiratory illness and influenza-like illness were 3.1 and 2.0 episodes per 100 person-weeks, respectively. A pathogen was detected in 96% of samples. The most commonly detected microbes were Haemophilus influenzae type B (62.7%) or non-type B (58.2%) and rhinovirus (22.4%). At baseline, bacterial colonization was high and included H. influenzae type B (82.3%), H. influenzae non-type B (31.5%), Klebsiella pneumoniae (14.6%), Staphylococcus aureus (8.5%), and Streptococcus pneumoniae (8.5%). At the end of follow-up, colonization with H. influenzae non-type B had increased to 74.1%, and S. pneumoniae to 33.6%. In the serology subset, the rate of influenza infection was 3.4 per 100 person-months; 58% of influenza infections resulted in clinical disease. Our study provides key data on the epidemiology and transmission of respiratory pathogens in tropical settings. Our results emphasize the need for improved infection prevention and control in military environments, given the high burden of illness and potential for intense transmission of respiratory pathogens.
Assuntos
Infecções por Haemophilus/epidemiologia , Influenza Humana/epidemiologia , Infecções por Klebsiella/epidemiologia , Infecções por Picornaviridae/epidemiologia , Pneumonia Pneumocócica/epidemiologia , Infecções Respiratórias/epidemiologia , Infecções Estafilocócicas/epidemiologia , Adolescente , Adulto , Infecções por Haemophilus/transmissão , Haemophilus influenzae tipo b/genética , Haemophilus influenzae tipo b/isolamento & purificação , Humanos , Incidência , Influenza Humana/transmissão , Infecções por Klebsiella/transmissão , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Masculino , Militares , Orthomyxoviridae/genética , Orthomyxoviridae/isolamento & purificação , Infecções por Picornaviridae/transmissão , Pneumonia Pneumocócica/transmissão , Reação em Cadeia da Polimerase , Estudos Prospectivos , Infecções Respiratórias/transmissão , Rhinovirus/genética , Rhinovirus/isolamento & purificação , Infecções Estafilocócicas/transmissão , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação , Tailândia/epidemiologiaRESUMO
Mycotic aneurysm is a rare but life-threatening disease that warrants an integrated therapeutic approach involving surgical intervention and prolonged antibiotic use. However, the causative organisms are often unidentified because antibiotics started empirically render blood and tissue cultures negative. Molecular diagnosis has been reported to be useful in such culture-negative cases. We report a case of a culture-negative mycotic aortic aneurysm due to Haemophilus influenzae, diagnosed by direct 16S rRNA polymerase chain reaction (PCR) and sequencing of the resected aneurysm tissue. PCR for serotype revealed type b, and PCR and sequencing of the ftsI gene revealed alterations in penicillin-binding protein 3, suggesting resistance to ampicillin. Multilocus sequence typing demonstrated that the isolate belonged to sequence type 54.
Assuntos
Aneurisma Infectado/microbiologia , Aneurisma Aórtico/microbiologia , Infecções por Haemophilus/microbiologia , Haemophilus influenzae tipo b/genética , Tipagem de Sequências Multilocus , Idoso , Resistência a Ampicilina/genética , Aneurisma Infectado/diagnóstico por imagem , Aneurisma Aórtico/diagnóstico por imagem , Bases de Dados de Ácidos Nucleicos , Infecções por Haemophilus/diagnóstico por imagem , Humanos , Masculino , Proteínas de Ligação às Penicilinas/genética , RNA Ribossômico 16S/genética , SorogrupoRESUMO
BACKGROUND: The aim of this study is to compare the diagnostic performance of the line probe assay (LPA) with conventional multiplex polymerase chain reaction (PCR) for Streptococcus pneumoniae as well as real-time PCR for Neisseria meningitidis and Haemophilus influenzae type b (Hib) in cerebrospinal fluid (CSF) samples from children during the multicenter national surveillance of bacterial meningitis between the years 2006 and 2009 in Turkey. RESULTS: During the study period 1460 subjects were enrolled and among them 841 (57%) met the criteria for probable bacterial meningitis. The mean age of subjects was 51 ± 47 months (range, 1-212 months). We performed the line probe assay in 751 (89%) CSF samples of 841 probable bacterial meningitis cases, of whom 431 (57%) were negative, 127 (17%) were positive for S. pneumoniae, 53 (7%) were positive for H. influenzae type b, and 41 (5%) were positive for N. meningitidis. The LPA was positive in 19 of 23 (82%) S. pneumoniae samples, 4 of 6 (67%) N. meningitidis samples and 2 of 2 (100%) Hib samples in CSF culture-positive cases. The specificity of the LPA for all of S. pneumoniae, H. influenzae type b, and N. meningitidis was 88% (95% CI: 85-91%), when using the standard PCR as a reference. The specificity of LPA for each of S. pneumoniae, H. influenzae type b, and N. meningitidis was 93% (95% CI: 89-95%), 96% (95% CI: 94-98%), and 99% (95% CI: 97-99%), respectively. For all of S. pneumoniae, H. influenzae type b and N. meningitidis the sensitivity of the LPA was 76% (95% CI: 70-82%) and for each of S. pneumoniae, H. influenzae type b and N. meningitidis was 72% (95% CI:63-79%), 88% (95% CI: 73-95%), and 81% (95% CI:67-92%), respectively. CONCLUSIONS: The LPA assay can be used to detect common bacterial meningitis pathogens in CSF samples, but the assay requires further improvement.
Assuntos
Bactérias/genética , Bactérias/isolamento & purificação , Técnicas de Tipagem Bacteriana/métodos , Líquido Cefalorraquidiano/microbiologia , Meningites Bacterianas/líquido cefalorraquidiano , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/microbiologia , Adolescente , Criança , Pré-Escolar , DNA Bacteriano , Feminino , Haemophilus influenzae tipo b/genética , Haemophilus influenzae tipo b/isolamento & purificação , Humanos , Lactente , Masculino , Meningite por Haemophilus/epidemiologia , Meningite por Haemophilus/microbiologia , Meningite Meningocócica/epidemiologia , Meningite Meningocócica/microbiologia , Meningite Pneumocócica/epidemiologia , Meningite Pneumocócica/microbiologia , Técnicas de Sonda Molecular , Reação em Cadeia da Polimerase Multiplex/métodos , Neisseria meningitidis/genética , Neisseria meningitidis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação , Turquia/epidemiologiaRESUMO
During 5 months in 2014, three Amish children in Missouri, USA, were diagnosed with invasive Haemophilus influenzae type b infection. Two were rural neighbors infected with a genetically similar rare strain, sequence type 45. One child had recently traveled, raising the possibility of maintenance of this strain among unvaccinated carriers in Amish communities.
Assuntos
Amish/psicologia , Infecções por Haemophilus/etnologia , Infecções por Haemophilus/epidemiologia , Haemophilus influenzae tipo b/patogenicidade , Pré-Escolar , Feminino , Infecções por Haemophilus/prevenção & controle , Infecções por Haemophilus/transmissão , Vacinas Anti-Haemophilus/administração & dosagem , Haemophilus influenzae tipo b/classificação , Haemophilus influenzae tipo b/genética , Haemophilus influenzae tipo b/isolamento & purificação , Humanos , Lactente , Masculino , Missouri/epidemiologia , Tipagem de Sequências Multilocus , Vacinação/psicologiaRESUMO
Haemophilus influenzae type b (Hib) causes acute bacterial meningitis (ABM) in children, with a mortality rate of about 3-6 % of the affected patients. ABM can lead to death during a period of hours to several days and, hence, rapid and early detection of the infection is crucial. Aptamers, the short single-stranded DNA or RNA with high affinity to target molecules, are selected by a high-flux screening technique known as in vitro screening and systematic evolution of ligands by exponential enrichment technology (SELEX). In this study, whole-cell SELEX was applied for the selection of target-specific aptamers with high affinity to Hib. ssDNA aptamers prepared by lambda exonuclease were incubated with the target cells (Hib). The aptameric binding rate to Hib was characterized for binding affinity after seven SELEX rounds by flow cytometry. The aptamers with higher binding affinity were cloned. Four of 68 aptamer clones were selected for sequencing. The dissociation constant (Kd) of the high-affinity aptamer clones 45 and 63 were 47.10 and 28.46 pM, respectively. These aptamers did not bind to other bacterial species, including the seven meningitis-causing bacteria. They showed distinct affinity to various H. influenzae strains only. These aptamers showed the highest affinity to Hib and the lowest affinity to H. influenzae type c and to other meningitis-causing bacteria. Clone 63 could detect Hib in patients' cerebrospinal fluid (CSF) samples at 60 colony-forming units (CFU)/mL. The results indicate applicability of the aptamers for rapid and early detection of infections brought about by Hib.
Assuntos
Aptâmeros de Nucleotídeos , Infecções por Haemophilus/diagnóstico , Infecções por Haemophilus/microbiologia , Haemophilus influenzae tipo b/genética , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/química , Citometria de Fluxo , Humanos , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
The majority of conjugate vaccines focus on inducing an antibody response to the polysaccharide antigen and the carrier protein is present primarily to induce a T-cell dependent response. In this study conjugates consisting of poly(ribosylribitolphosphate) (PRP) purified from Haemophilus influenzae Type b bound to Hepatitis B virus surface antigen (HBsAg) virus like particles were prepared with the aim of inducing an antibody response to not only the PRP but also the HBsAg. A conjugate consisting of PRP bound to HBsAg via an adipic acid dihydrazide (ADH) spacer induced strong IgG antibodies to both the PRP and HBsAg. When conjugation was performed without the ADH spacer the induction of an anti-PRP response was equivalent to that seen by conjugate with the ADH spacer, however, a negligible anti-HBsAg response was induced. For comparison, PRP was conjugated to diphtheria toxoid (DT) and Vi polysaccharide purified from Salmonella Typhi conjugated to HBsAg both using an ADH spacer. The PRPAH-DT conjugate induced strong anti-PRP and anti-DT responses, the Vi-AHHBsAg conjugate induced a good anti-HBsAg response but not as strong as that induced by the PRPAH-HBsAg conjugate. This study demonstrated that in mice it was possible to induce robust antibody responses to both polysaccharide and carrier protein provided the conjugate has certain physico-chemical properties. A PRPAH-HBsAg conjugate with the capacity to induce anti-PRP and anti-HBsAg responses could be incorporated into a multivalent pediatric vaccine and simplify formulation of such a vaccine.
Assuntos
Vacinas Anti-Haemophilus/imunologia , Haemophilus influenzae tipo b/imunologia , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Polissacarídeos/imunologia , Vacinas Conjugadas/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Toxoide Diftérico/genética , Toxoide Diftérico/imunologia , Vacina contra Difteria, Tétano e Coqueluche/administração & dosagem , Vacina contra Difteria, Tétano e Coqueluche/imunologia , Haemophilus influenzae tipo b/química , Haemophilus influenzae tipo b/genética , Antígenos de Superfície da Hepatite B/química , Imunoglobulina G/imunologia , Camundongos , Polissacarídeos/genética , Polissacarídeos/isolamento & purificação , Salmonella typhi/genética , Salmonella typhi/imunologia , Vacinas Conjugadas/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/imunologiaRESUMO
The occurrence and significance of Haemophilus spp. isolated from the genitourinary tract are not well known. Herein, we describe the clinical significance and characteristics of Haemophilus influenzae type b genogroup strains isolated from genitourinary tract specimens from an adult male veteran patient population and, in particular, their associations with prostatitis and epididymitis.
Assuntos
Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/urina , Haemophilus influenzae tipo b/genética , Haemophilus influenzae tipo b/isolamento & purificação , Adulto , Epididimite/microbiologia , Epididimite/urina , Genótipo , Humanos , Masculino , Testes de Sensibilidade Microbiana/métodos , Prostatite/microbiologia , Prostatite/urinaRESUMO
BACKGROUND: Dried blood spot (DBS) is a reliable blood collection method for storing samples at room temperature and easily transporting them. We have previously validated a Real-Time PCR for detection of Streptococcus pneumoniae in DBS. The objective of this study was to apply this methodology for the diagnosis of S. pneumoniae and Haemophilus influenzae b (Hib) in DBS samples of children with pneumonia admitted to two hospitals in Mozambique and Morocco. METHODS: Ply and wzg genes of S. pneumoniae and bexA gene of Hib, were used as targets of Real-Time PCR. 329 DBS samples of children hospitalized with clinical diagnosis of pneumonia were tested. RESULTS: Real-Time PCR in DBS allowed for a significant increase in microbiological diagnosis of S. pneumoniae and Hib. When performing blood bacterial culture, only ten isolates of S. pneumoniae and none of Hib were detected (3·0% positivity rate, IC95% 1·4-5·5%). Real-Time PCR from DBS samples increased the detection yield by 4x fold, as 30 S. pneumoniae and 11 Hib cases were detected (12·4% positivity rate, IC95% 9·0-16·5%; P<0·001). CONCLUSION: Real-Time PCR applied in DBS may be a valuable tool for improving diagnosis and surveillance of pneumonia caused by S. pneumoniae or Hib in developing countries.
Assuntos
Infecções por Haemophilus/microbiologia , Haemophilus influenzae tipo b/genética , Pneumonia Pneumocócica/microbiologia , Streptococcus pneumoniae/genética , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Coleta de Amostras Sanguíneas/métodos , Criança , Pré-Escolar , DNA Bacteriano/sangue , DNA Bacteriano/genética , Países em Desenvolvimento , Feminino , Infecções por Haemophilus/sangue , Infecções por Haemophilus/diagnóstico , Haemophilus influenzae tipo b/isolamento & purificação , Hospitais , Humanos , Lactente , Masculino , Marrocos , Moçambique , Pneumonia Pneumocócica/sangue , Pneumonia Pneumocócica/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Streptococcus pneumoniae/isolamento & purificação , Estreptolisinas/genéticaRESUMO
OBJECTIVE: To express the recombinant D protein in prokaryotic expression system solubly and make preparation for producing D-carrier conjugate vaccine next step. METHODS: The hpd gene fragment removed of signal peptide from genomic DNA of Hib CMCC was inserted into pET43. 1a. The recombinant plasmid was transformed to competent E. coli BL21 (DE3) for expression under induction of IPTG. The expressed recombination protein was precipitated with ammonium sulfate, purified by DEAE anion exchange column chromatography and identified for reactogenicity by Western Blot. RESULTS: The expressed recombination protein, in a soluble form, constained about 50% of total somatic protein and showed specific reaction with the HIB antisera after preliminary purification. CONCLUSION: The D protein recombined expression plasmid was constructed successfully and expressed D protein in prokaryotic cells in a solube form.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Haemophilus influenzae tipo b/genética , Imunoglobulina D/genética , Lipoproteínas/genética , Western Blotting , Escherichia coli/genética , Plasmídeos , Proteínas Recombinantes/biossíntese , SolubilidadeRESUMO
A real-time polymerase chain reaction assay that uses degenerate primers and a dual-labelled probe was developed to detect the bexA gene of Haemophilus influenzae, including those belonging to non-b serotypes as well as clonal division II strains. This assay is sensitive and specific, detecting 20 copies of the gene, but negative with a variety of bacteria associated with meningitis and bacteremia or septicemia.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Infecções por Haemophilus/diagnóstico , Haemophilus influenzae/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Líquido Cefalorraquidiano/microbiologia , Primers do DNA/genética , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/classificação , Haemophilus influenzae/genética , Haemophilus influenzae tipo b/genética , Haemophilus influenzae tipo b/isolamento & purificação , Humanos , Lactente , Meningite/microbiologia , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
We report a rare case of infection by two different types of Haemophilus influenzae strains in a child who received only one dose of the H. influenzae serotype b (Hib) conjugate vaccine (DTwP+Hib). The strains were recovered from blood and cerebrospinal fluid (CSF) and were phenotypically identified as Hib and non-typable H. influenzae, respectively, after serological tests. The two strains were characterized by PCR capsular typing, multilocus sequence typing and PFGE. Our results suggest that the infection was caused by the bloodstream invasion by a single Hib strain, followed by the diffusion of the bacteria across the blood-brain barrier and into the CSF. The strain recovered from the CSF, however, was identified as a capsule-deficient type mutant (b(-)) strain. Despite the high efficacy of the Hib conjugate vaccine, the increase in the numbers of strains able to escape the immune system of the vaccinated population advocates continued surveillance.
Assuntos
Cápsulas Bacterianas/genética , Infecções por Haemophilus/diagnóstico , Infecções por Haemophilus/patologia , Haemophilus influenzae tipo b/isolamento & purificação , Mutação , Brasil , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Genótipo , Infecções por Haemophilus/imunologia , Infecções por Haemophilus/microbiologia , Vacinas Anti-Haemophilus/administração & dosagem , Vacinas Anti-Haemophilus/imunologia , Haemophilus influenzae tipo b/genética , Haemophilus influenzae tipo b/imunologia , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
In Japan, ß-lactamase-nonproducing, ampicillin-resistant organisms have been evident among Haemophilus influenzae type b (Hib) isolates since 2000, when no appropriate vaccine had been approved. We therefore performed molecular analysis of agents causing H. influenzae meningitis nationwide over the following 10 years. Some 285 institutions have participated in surveillance since 2000. The capsular type and resistance genes of 1,353 isolates and 23 cerebrospinal fluid samples from pediatric patients with meningitis we had received from 2000 to 2011 were analyzed by polymerase chain reaction. Blood and spinal fluid test results obtained when patients were admitted were examined for correlation with outcomes. Hib was found in 98.9 % of isolates. We received more than 100 Hib isolates per year until vaccination began in December 2008, when these isolates decreased, especially since establishment of a special fund to promote vaccination in November 2010. Decreased incidence among infants 7 months to 2 years old has been particularly notable. However, the rate of ampicillin-resistant organisms has increased to more than 60 % of all isolates since 2009. We received 587 replies to a questionnaire concerning outcomes, indicating 2 % mortality and 17.7 % serious morbidity. Age of 6 months or younger and presence of disseminated intravascular coagulation at admission were related to an unfavorable outcome (p < 0.05), but ampicillin resistance was not. Combination therapy with third-generation cephem and carbapenem agents was used initially for 72 % of patients. Routine immunization can prevent Hib meningitis in children.