RESUMO
Laboratories play critical roles in bacterial meningitis disease surveillance in the African meningitis belt, where the highest global burden of meningitis exists. Reinforcement of laboratory capacity ensures rapid detection of meningitis cases and outbreaks and a public health response that is timely, specific, and appropriate. Since 2008, joint efforts to strengthen laboratory capacity by multiple partners, including MenAfriNet, beginning in 2014, have been made in countries within and beyond the meningitis belt. Over the course of 10 years, national reference laboratories were supported in 5 strategically targeted areas: specimen transport systems, laboratory procurement systems, laboratory diagnosis, quality management, and laboratory workforce with substantial gains made in each of these areas. To support the initiative to eliminate meningitis by 2030, continued efforts are needed to strengthen laboratory systems.
Assuntos
Técnicas de Laboratório Clínico , Laboratórios , Meningites Bacterianas/epidemiologia , África Subsaariana/epidemiologia , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Surtos de Doenças , Mão de Obra em Saúde , História do Século XXI , Humanos , Laboratórios/organização & administração , Laboratórios/provisão & distribuição , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/história , Meningites Bacterianas/microbiologia , Vigilância da População , Qualidade da Assistência à SaúdeRESUMO
Haemophilus haemolyticus has been recently discovered to have the potential to cause invasive disease. It is closely related to nontypeable Haemophilus influenzae (NT H. influenzae). NT H. influenzae and H. haemolyticus are often misidentified because none of the existing tests targeting the known phenotypes of H. haemolyticus are able to specifically identify H. haemolyticus Through comparative genomic analysis of H. haemolyticus and NT H. influenzae, we identified genes unique to H. haemolyticus that can be used as targets for the identification of H. haemolyticus A real-time PCR targeting purT (encoding phosphoribosylglycinamide formyltransferase 2 in the purine synthesis pathway) was developed and evaluated. The lower limit of detection was 40 genomes/PCR; the sensitivity and specificity in detecting H. haemolyticus were 98.9% and 97%, respectively. To improve the discrimination of H. haemolyticus and NT H. influenzae, a testing scheme combining two targets (H. haemolyticus purT and H. influenzae hpd, encoding protein D lipoprotein) was also evaluated and showed 96.7% sensitivity and 98.2% specificity for the identification of H. haemolyticus and 92.8% sensitivity and 100% specificity for the identification of H. influenzae, respectively. The dual-target testing scheme can be used for the diagnosis and surveillance of infection and disease caused by H. haemolyticus and NT H. influenzae.
Assuntos
Hibridização Genômica Comparativa/métodos , Infecções por Haemophilus/diagnóstico , Haemophilus influenzae/classificação , Haemophilus influenzae/genética , Lipoproteínas/genética , Fosforribosilglicinamido Formiltransferase/genética , Sequência de Bases , DNA Bacteriano/genética , Genoma Bacteriano/genética , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/isolamento & purificação , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
Neisseria meningitidis serogroup B (MnB) is a leading cause of bacterial meningitis; however, MnB is most commonly associated with asymptomatic carriage in the nasopharyngeal cavity, as opposed to the disease state. Two vaccines are now licensed for the prevention of MnB disease; a possible additional benefit of these vaccines could be to protect against disease indirectly by disrupting nasopharyngeal carriage (e.g., herd protection). To investigate this possibility, accurate diagnostic approaches to characterize MnB carriage isolates are required. In contrast to invasive meningococcal disease (IMD) isolates, which can be readily serogrouped, carriage isolates often lack capsule expression, making standard phenotypic assays unsuitable for strain characterization. Several antibody-based methods were evaluated for their abilities to serogroup isolates and were compared with two genotyping methods (real-time PCR [rt-PCR] and whole-genome sequencing [WGS]) to identify which approach would most accurately ascertain the polysaccharide groups associated with carriage isolates. WGS and rt-PCR were in agreement for 99% of IMD isolates, including those with coding sequences for MnB, MnC, MnW, and MnY, and the phenotypic methods correctly identified serogroups for 69 to 98% of IMD isolates. In contrast, only 47% of carriage isolates were groupable by genotypic methods, due to mutations within the capsule operon; of the isolates identified by genotypic methods, ≤43% were serogroupable with any of the phenotypic methods tested. These observations highlight the difficulties in the serogrouping and capsular genogrouping of meningococcal carriage isolates. Based on our findings, WGS is the most suitable approach for the characterization of meningococcal carriage isolates.
Assuntos
Cápsulas Bacterianas/classificação , Portador Sadio/microbiologia , Técnicas de Genotipagem/métodos , Neisseria meningitidis/classificação , Infecções por Neisseriaceae/microbiologia , Sorotipagem/métodos , Adolescente , Adulto , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/imunologia , Estudos Epidemiológicos , Feminino , Humanos , Masculino , Neisseria meningitidis/genética , Neisseria meningitidis/imunologia , Adulto JovemRESUMO
BACKGROUND: Meningococcal conjugate vaccines against serogroups A, C, W, and Y (MenACWY) are recommended for routine use in adolescents aged 11-18 years. The impact of these vaccines on the meningococcal population structure in the United States have yet to be evaluated. METHODS: Meningococcal isolates recovered during 2006-2010 (ie, after introduction of MenACWY) collected through Active Bacterial Core surveillance (ABCs) were characterized; serogroup distribution and molecular features of these isolates were compared to previously published data on ABCs isolates recovered from 2000 to 2005 (ie, before introduction of MenACWY). P values were generated using χ(2) statistics and exact methods. RESULTS: There was a significant change (P < .05) in serogroup distribution among all age groups between the 2 periods. A small proportion of isolates showed evidence of capsular switching in both periods. Between the 2 periods, significant changes were observed in the distribution of porin A, ferric enterobactin transport, and strain genotypes among vaccine and nonvaccine serogroups. CONCLUSIONS: The population structure of US meningococcal isolates is dynamic; some changes occurred over time, but the basic structure remained. Vaccine-induced serogroup replacement was not observed, although a small proportion of isolates had undergone capsule switching, possibly driven by non-vaccine-mediated selection. Changes in the distribution of molecular features are likely due to horizontal gene transfer and changes in serogroup distribution.
Assuntos
Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/microbiologia , Vacinas Meningocócicas/administração & dosagem , Neisseria meningitidis/classificação , Neisseria meningitidis/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Infecções Meningocócicas/prevenção & controle , Pessoa de Meia-Idade , Neisseria meningitidis/genética , Neisseria meningitidis/imunologia , Sorogrupo , Estados Unidos , Vacinas Conjugadas/administração & dosagem , Adulto JovemRESUMO
BACKGROUND: Meningococcal disease incidence in the United States is at an all-time low. In a previous study of Georgia high school students, meningococcal carriage prevalence was 7%. The purpose of this study was to measure the impact of a meningococcal conjugate vaccine on serogroup Y meningococcal carriage and to define the dynamics of carriage in high school students. METHODS: This was a prospective cohort study at 8 high schools, 4 each in Maryland and Georgia, during a school year. Students at participating schools received quadrivalent meningococcal conjugate vaccine that uses diphtheria toxoid as the protein carrier (MCV4-DT). In each state, 2 high schools were randomly assigned for MCV4-DT receipt by students at the beginning of the study, and 2 were randomly assigned for MCV4-DT receipt at the end. Oropharyngeal swab cultures for meningococcal carriage were performed 3 times during the school year. RESULTS: Among 3311 students, the prevalence of meningococcal carriage was 3.21%-4.01%. Phenotypically nongroupable strains accounted for 88% of carriage isolates. There were only 5 observed acquisitions of serogroup Y strains during the study; therefore, the impact of MCV4-DT on meningococcal carriage could not be determined. CONCLUSIONS: Meningococcal carriage rates in US high school students were lower than expected, and the vast majority of strains did not express capsule. These findings may help explain the historically low incidence of meningococcal disease in the United States.
Assuntos
Portador Sadio/epidemiologia , Portador Sadio/microbiologia , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/microbiologia , Neisseria meningitidis/imunologia , Estudantes/estatística & dados numéricos , Adolescente , Adulto , Portador Sadio/imunologia , Portador Sadio/prevenção & controle , Feminino , Georgia/epidemiologia , Humanos , Masculino , Maryland/epidemiologia , Infecções Meningocócicas/imunologia , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/imunologia , Neisseria meningitidis/classificação , Estudos Prospectivos , Fatores de Risco , Adulto JovemRESUMO
BACKGROUND: During the first introduction of a group A meningococcal vaccine (PsA-TT) in 2010-2011 and its rollout from 2011 to 2013, >150 million eligible people, representing 12 hyperendemic meningitis countries, have been vaccinated. METHODS: The new vaccine effectiveness evaluation framework was established by the World Health Organization and partners. Meningitis case-based surveillance was strengthened in PsA-TT first-introducer countries, and several evaluation studies were conducted to estimate the vaccination coverage and to measure the impact of vaccine introduction on meningococcal carriage and disease incidence. RESULTS: PsA-TT implementation achieved high vaccination coverage, and results from studies conducted showed significant decrease of disease incidence as well as significant reduction of oropharyngeal carriage of group A meningococci in vaccinated and unvaccinated individuals, demonstrating the vaccine's ability to generate herd protection and prevent group A epidemics. CONCLUSIONS: Lessons learned from this experience provide useful insights in how to guide and better prepare for future new vaccine introductions in resource-limited settings.
Assuntos
Portador Sadio/epidemiologia , Portador Sadio/prevenção & controle , Transmissão de Doença Infecciosa/prevenção & controle , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/administração & dosagem , Vacinas Meningocócicas/imunologia , Adolescente , Adulto , África/epidemiologia , Criança , Pré-Escolar , Feminino , Humanos , Incidência , Lactente , Masculino , Resultado do Tratamento , Adulto JovemRESUMO
During September 2006-December 2009, we conducted active population and sentinel laboratory-based surveillance for bacterial meningitis pathogens, including Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae type b, in 4 China prefectures. We identified 7,876 acute meningitis and encephalitis syndrome cases, including 6,388 among prefecture residents. A total of 833 resident cases from sentinel hospitals met the World Health Organization case definition for probable bacterial meningitis; 339 of these cases were among children <5 years of age. Laboratory testing confirmed bacterial meningitis in 74 of 3,391 tested cases. The estimated annual incidence (per 100,000 population) of probable bacterial meningitis ranged from 1.84 to 2.93 for the entire population and from 6.95 to 22.30 for children <5 years old. Active surveillance with laboratory confirmation has provided a population-based estimate of the number of probable bacterial meningitis cases in China, but more complete laboratory testing is needed to better define the epidemiology of the disease in this country.
Assuntos
Meningites Bacterianas/epidemiologia , Vigilância da População , Criança , Pré-Escolar , China/epidemiologia , Geografia Médica , Humanos , Incidência , Lactente , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/microbiologia , Estações do Ano , Vigilância de Evento SentinelaRESUMO
In 2010, Burkina Faso became the first country to introduce meningococcal serogroup A conjugate vaccine (PsA-TT). During 2012, Burkina Faso reported increases in Neisseria meningitidis serogroup W, raising questions about whether these cases were a natural increase in disease or resulted from serogroup replacement after PsA-TT introduction. We analyzed national surveillance data to describe the epidemiology of serogroup W and genotyped 61 serogroup W isolates. In 2012, a total of 5,807 meningitis cases were reported through enhanced surveillance, of which 2,353 (41%) were laboratory confirmed. The predominant organism identified was N. meningitidis serogroup W (62%), and all serogroup W isolates characterized belonged to clonal complex 11. Although additional years of data are needed before we can understand the epidemiology of serogroup W after PsA-TT introduction, these data suggest that serogroup W will remain a major cause of sporadic disease and has epidemic potential, underscoring the need to maintain high-quality case-based meningitis surveillance after PsA-TT introduction.
Assuntos
Meningite Meningocócica/epidemiologia , Neisseria meningitidis/classificação , Sorogrupo , Adolescente , Burkina Faso/epidemiologia , Criança , Pré-Escolar , Genótipo , História do Século XXI , Humanos , Incidência , Lactente , Recém-Nascido , Meningite Meningocócica/história , Neisseria meningitidis/genética , Vigilância da População , Adulto JovemRESUMO
Occupationally acquired meningococcal disease is rare. Adherence to recommendations for safe handling of Neisseria meningitidis in the laboratory greatly reduces the risk for transmission to laboratory workers. A California microbiologist developed fatal serogroup B meningococcal disease after working with N. meningitidis patient isolates in a research laboratory (laboratory A). The California Department of Public Health (CDPH), the local health department, the California Division of Occupational Safety and Health (CalOSHA), and the federal Occupational Safety and Health Administration (OSHA) collaborated on an investigation of laboratory A, which revealed several breaches in recommended laboratory practice for safe handling of N. meningitidis, including manipulating cultures on the bench top. Additionally, laboratory workers had not been offered meningococcal vaccine in accordance with Advisory Committee on Immunization Practices (ACIP) recommendations and CalOSHA Aerosol Transmissible Diseases Standard requirements. In accordance with OSHA and CalOSHA regulations, laboratory staff members must receive laboratory biosafety training and use appropriate personal protective equipment, and those who routinely work with N. meningitidis isolates should receive meningococcal vaccine.
Assuntos
Infecções Meningocócicas/diagnóstico , Neisseria meningitidis/isolamento & purificação , Doenças Profissionais/diagnóstico , Adulto , California , Evolução Fatal , Humanos , Laboratórios , MasculinoRESUMO
BACKGROUND: College students living in residential halls are at increased risk of meningococcal disease. Unlike that for serogroups prevented by quadrivalent meningococcal vaccines, public health response to outbreaks of serogroup B meningococcal disease is limited by lack of a US licensed vaccine. METHODS: In March 2010, we investigated a prolonged outbreak of serogroup B disease associated with a university. In addition to case ascertainment, molecular typing of isolates was performed to characterize the outbreak. We conducted a matched case-control study to examine risk factors for serogroup B disease. Five controls per case, matched by college year, were randomly selected. Participants completed a risk factor questionnaire. Data were analyzed using conditional logistic regression. RESULTS: Between January 2008 and November 2010, we identified 13 meningococcal disease cases (7 confirmed, 4 probable, and 2 suspected) involving 10 university students and 3 university-linked persons. One student died. Ten cases were determined to be serogroup B. Isolates from 6 confirmed cases had an indistinguishable pulsed-field gel electrophoresis pattern and belonged to sequence type 269, clonal complex 269. Factors significantly associated with disease were Greek society membership (matched odds ratio [mOR], 15.0; P = .03), >1 kissing partner (mOR, 13.66; P = .03), and attending bars (mOR, 8.06; P = .04). CONCLUSIONS: The outbreak was associated with a novel serogroup B strain (CC269) and risk factors were indicative of increased social mixing. Control measures were appropriate but limited by lack of vaccine. Understanding serogroup B transmission in college and other settings will help inform use of serogroup B vaccines currently under consideration for licensure.
Assuntos
Surtos de Doenças , Meningite Meningocócica/epidemiologia , Meningite Meningocócica/microbiologia , Neisseria meningitidis Sorogrupo B/classificação , Neisseria meningitidis Sorogrupo B/isolamento & purificação , Adolescente , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Fatores de Risco , Sorotipagem , Inquéritos e Questionários , Estados Unidos/epidemiologia , Universidades , Adulto JovemRESUMO
BACKGROUND: Although cerebrospinal fluid (CSF) culture is the diagnostic reference standard for bacterial meningitis, its sensitivity is limited, particularly when antibiotics were previously administered. CSF Gram staining and real-time PCR are theoretically less affected by antibiotics; however, it is difficult to evaluate these tests with an imperfect reference standard. METHODS AND FINDINGS: CSF from patients with suspected meningitis from Salvador, Brazil were tested with culture, Gram stain, and real-time PCR using S. pneumoniae, N. meningitidis, and H. influenzae specific primers and probes. An antibiotic detection disk bioassay was used to test for the presence of antibiotic activity in CSF. The diagnostic accuracy of tests were evaluated using multiple methods, including direct evaluation of Gram stain and real-time PCR against CSF culture, evaluation of real-time PCR against a composite reference standard, and latent class analysis modeling to evaluate all three tests simultaneously. RESULTS: Among 451 CSF specimens, 80 (17.7%) had culture isolation of one of the three pathogens (40 S. pneumoniae, 36 N. meningitidis, and 4 H. influenzae), and 113 (25.1%) were real-time PCR positive (51 S. pneumoniae, 57 N. meningitidis, and 5 H. influenzae). Compared to culture, real-time PCR sensitivity and specificity were 95.0% and 90.0%, respectively. In a latent class analysis model, the sensitivity and specificity estimates were: culture, 81.3% and 99.7%; Gram stain, 98.2% and 98.7%; and real-time PCR, 95.7% and 94.3%, respectively. Gram stain and real-time PCR sensitivity did not change significantly when there was antibiotic activity in the CSF. CONCLUSION: Real-time PCR and Gram stain were highly accurate in diagnosing meningitis caused by S. pneumoniae, N. meningitidis, and H. influenzae, though there were few cases of H. influenzae. Furthermore, real-time PCR and Gram staining were less affected by antibiotic presence and might be useful when antibiotics were previously administered. Gram staining, which is inexpensive and commonly available, should be encouraged in all clinical settings.
Assuntos
Haemophilus influenzae/genética , Meningites Bacterianas/diagnóstico , Neisseria meningitidis/genética , Streptococcus pneumoniae/genética , Adolescente , Adulto , Idoso , Líquido Cefalorraquidiano/microbiologia , Criança , Pré-Escolar , Violeta Genciana , Haemophilus influenzae/isolamento & purificação , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Neisseria meningitidis/isolamento & purificação , Fenazinas , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Streptococcus pneumoniae/isolamento & purificação , Adulto JovemRESUMO
Containment strategies for outbreaks of invasive Neisseria meningitidis disease are informed by serogroup assays that characterize the polysaccharide capsule. We sought to uncover the genomic basis of conflicting serogroup assay results for an isolate (M16917) from a patient with acute meningococcal disease. To this end, we characterized the complete genome sequence of the M16917 isolate and performed a variety of comparative sequence analyses against N. meningitidis reference genome sequences of known serogroups. Multilocus sequence typing and whole-genome sequence comparison revealed that M16917 is a member of the ST-11 sequence group, which is most often associated with serogroup C. However, sequence similarity comparisons and phylogenetic analysis showed that the serogroup diagnostic capsule polymerase gene (synD) of M16917 belongs to serogroup B. These results suggest that a capsule-switching event occurred based on homologous recombination at or around the capsule locus of M16917. Detailed analysis of this locus uncovered the locations of recombination breakpoints in the M16917 genome sequence, which led to the introduction of an â¼2-kb serogroup B sequence cassette into the serogroup C genomic background. Since there is no currently available vaccine for serogroup B strains of N. meningitidis, this kind capsule-switching event could have public health relevance as a vaccine escape mutant.
Assuntos
Genoma Bacteriano , Neisseria meningitidis/classificação , Neisseria meningitidis/genética , Polissacarídeos Bacterianos/metabolismo , Testes de Aglutinação , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Infecções Meningocócicas/microbiologia , Dados de Sequência Molecular , Neisseria meningitidis/imunologia , Neisseria meningitidis/isolamento & purificação , Filogenia , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/imunologia , Análise de Sequência de DNA , Homologia de Sequência , SorotipagemRESUMO
We report on three cases of meningococcal disease caused by ciprofloxacin-resistant Neisseria meningitidis, one in North Dakota and two in Minnesota. The cases were caused by the same serogroup B strain. To assess local carriage of resistant N. meningitidis, we conducted a pharyngeal-carriage survey and isolated the resistant strain from one asymptomatic carrier. Sequencing of the gene encoding subunit A of DNA gyrase (gyrA) revealed a mutation associated with fluoroquinolone resistance and suggests that the resistance was acquired by means of horizontal gene transfer with the commensal N. lactamica. In susceptibility testing of invasive N. meningitidis isolates from the Active Bacterial Core surveillance system between January 2007 and January 2008, an additional ciprofloxacin-resistant isolate was found, in this case from California. Ciprofloxacin-resistant N. meningitidis has emerged in North America.
Assuntos
Anti-Infecciosos/uso terapêutico , Ciprofloxacina/uso terapêutico , Farmacorresistência Bacteriana/genética , Infecções Meningocócicas/tratamento farmacológico , Neisseria meningitidis/genética , Mutação Puntual , Adolescente , Adulto , Idoso , Sequência de Bases , Portador Sadio/microbiologia , Humanos , Lactente , Infecções Meningocócicas/microbiologia , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Neisseria meningitidis/classificação , Neisseria meningitidis/efeitos dos fármacos , Neisseria meningitidis/isolamento & purificação , Faringe/microbiologia , Estados Unidos , Adulto JovemRESUMO
We report seven cases of Haemophilus haemolyticus invasive disease detected in the United States, which were previously misidentified as nontypeable Haemophilus influenzae. All cases had different symptoms and presentations. Our study suggests that a testing scheme that includes reliable PCR assays and standard microbiological methods should be used in order to improve H. haemolyticus identification.
Assuntos
Infecções por Haemophilus/diagnóstico , Infecções por Haemophilus/microbiologia , Haemophilus/classificação , Haemophilus/isolamento & purificação , Idoso , Técnicas Bacteriológicas/métodos , Criança , DNA Bacteriano/química , DNA Bacteriano/genética , Feminino , Infecções por Haemophilus/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA , Estados Unidos , Adulto JovemRESUMO
PCR detecting the protein D (hpd) and fuculose kinase (fucK) genes showed high sensitivity and specificity for identifying Haemophilus influenzae and differentiating it from H. haemolyticus. Phylogenetic analysis using the 16S rRNA gene demonstrated two distinct groups for H. influenzae and H. haemolyticus.
Assuntos
Marcadores Genéticos , Haemophilus influenzae/genética , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Imunoglobulina D/genética , Lipoproteínas/genética , Tipagem Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Filogenia , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , RNA Ribossômico 16S/genéticaRESUMO
Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA and serogroup-specific genes in the cap locus for N. meningitidis serogroups A, B, C, W135, X, and Y. However, the assay sensitivity for serogroups B, W135, and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays for serogroup B synD, W135 synG, and Y synF showed 100% specificity for detecting N. meningitidis species, with high sensitivity (serogroup B synD, 99% [75/76]; W135 synG, 97% [38/39]; and Y synF, 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup B synD, W135 synG, and Y synF assays, respectively, a significant improvement compared to results for the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of (i) N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA (NHS assay); (ii) N. meningitidis serogroups A, W135, and X (AWX assay); and (iii) N. meningitidis serogroups B, C, and Y (BCY assay). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for the singleplex assay. Pairwise comparison of real-time PCR between multiplex and singleplex assays showed that cycle threshold values of the multiplex assay were similar to those for the singleplex assay. There were no substantial differences in sensitivity and specificity between these multiplex and singleplex real-time PCR assays.
Assuntos
Haemophilus influenzae/isolamento & purificação , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/microbiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Neisseria meningitidis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Streptococcus pneumoniae/isolamento & purificação , Haemophilus influenzae/genética , Humanos , Neisseria meningitidis/classificação , Neisseria meningitidis/genética , Sensibilidade e Especificidade , Streptococcus pneumoniae/genéticaRESUMO
Neisseria meningitidis is one of the main agents of bacterial meningitis, causing substantial morbidity and mortality worldwide. However, most of the time N. meningitidis is carried as a commensal not associated with invasive disease. The genomic basis of the difference between disease-associated and carried isolates of N. meningitidis may provide critical insight into mechanisms of virulence, yet it has remained elusive. Here, we have taken a comparative genomics approach to interrogate the difference between disease-associated and carried isolates of N. meningitidis at the level of individual nucleotide variations (i.e., single nucleotide polymorphisms [SNPs]). We aligned complete genome sequences of 8 disease-associated and 4 carried isolates of N. meningitidis to search for SNPs that show mutually exclusive patterns of variation between the two groups. We found 63 SNPs that distinguish the 8 disease-associated genomes from the 4 carried genomes of N. meningitidis, which is far more than can be expected by chance alone given the level of nucleotide variation among the genomes. The putative list of SNPs that discriminate between disease-associated and carriage genomes may be expected to change with increased sampling or changes in the identities of the isolates being compared. Nevertheless, we show that these discriminating SNPs are more likely to reflect phenotypic differences than shared evolutionary history. Discriminating SNPs were mapped to genes, and the functions of the genes were evaluated for possible connections to virulence mechanisms. A number of overrepresented functional categories related to virulence were uncovered among SNP-associated genes, including genes related to the category "symbiosis, encompassing mutualism through parasitism."
Assuntos
Portador Sadio/microbiologia , Genoma Bacteriano , Infecções Meningocócicas/microbiologia , Neisseria meningitidis/genética , Polimorfismo de Nucleotídeo Único , Sequência de Bases , Variação Genética , Humanos , Dados de Sequência Molecular , Neisseria meningitidis/classificação , Neisseria meningitidis/isolamento & purificação , Neisseria meningitidis/patogenicidade , Filogenia , VirulênciaRESUMO
We report the first whole-genome sequences for five strains, two carried and three pathogenic, of the emerging pathogen Haemophilus haemolyticus. Preliminary analyses indicate that these genome sequences encode markers that distinguish H. haemolyticus from its closest Haemophilus relatives and provide clues to the identity of its virulence factors.
Assuntos
Genoma Bacteriano , Infecções por Haemophilus/microbiologia , Haemophilus/genética , Haemophilus/isolamento & purificação , Sequência de Bases , Haemophilus/classificação , Humanos , Dados de Sequência MolecularRESUMO
BACKGROUND: With the introduction of Haemophilus influenzae serotype b (Hib) conjugate vaccines, there has been a dramatic reduction of Hib disease in young children and the epidemiological trends of invasive H. influenzae have shifted. METHODS: Data were collected from active surveillance for invasive H. influenzae disease conducted through Active Bacterial Core surveillance sites during 1989-2008. RESULTS: During 1999-2008, the estimated mean annual incidence of H. influenzae infection was 1.62 cases per 100 000 population; 15.3% of cases were fatal. Incidence was higher among adults aged ≥65 years, compared with other age groups. The largest burden of disease among children aged <5 years was in infants aged <1 year; many of these cases occurred during the first month of life in preterm or low-birth weight infants. An estimated 10% of the total burden of disease among children aged <5 years occurred in American Indian and Alaska Native children. During 1989-2008, 7559 cases of H. influenzae disease were reported from Active Bacterial Core surveillance sites. Small increases in the incidence of serotypes a, e, and f were observed during 1989-2008. The largest of these increases was in serotype f and was primarily among adults aged ≥18 years. CONCLUSIONS: Since the introduction of Hib conjugate vaccines, the incidence of invasive disease caused by H. influenzae in the United States has decreased dramatically; however, a considerable burden of non-Hib disease is still present in the oldest and youngest age groups. There is no evidence of substantial replacement disease with non-b serotypes in young children in the United States.
Assuntos
Infecções por Haemophilus/epidemiologia , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/classificação , Haemophilus influenzae/isolamento & purificação , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Etnicidade , Feminino , Infecções por Haemophilus/mortalidade , Vacinas Anti-Haemophilus/administração & dosagem , Vacinas Anti-Haemophilus/imunologia , Humanos , Incidência , Lactente , Masculino , Pessoa de Meia-Idade , Sorotipagem , Estados Unidos/epidemiologia , Adulto JovemRESUMO
The large and growing number of viral and bacterial pathogens responsible for respiratory infections poses a challenge for laboratories seeking to provide rapid and comprehensive pathogen identification. We evaluated a novel application of the TaqMan low-density array (TLDA) cards for real-time PCR detection of 21 respiratory-pathogen targets. The performance of the TLDA was compared to that of individual real-time PCR (IRTP) assays with the same primers and probes using (i) nucleic acids extracted from the 21 pathogen strains and 66 closely related viruses and bacteria and (ii) 292 clinical respiratory specimens. With spiked samples, TLDA cards were about 10-fold less sensitive than IRTP assays. By using 292 clinical specimens to generate 2,238 paired individual assays, the TLDA card exhibited 89% sensitivity (95% confidence interval [CI], 86 to 92%; range per target, 47 to 100%) and 98% specificity (95% CI, 97 to 99%; range per target, 85 to 100%) overall compared to IRTP assays as the gold standard with a threshold cycle (C(T)) cutoff of 43. The TLDA card approach offers promise for rapid and simultaneous identification of multiple respiratory pathogens for outbreak investigations and disease surveillance.