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1.
J Clin Microbiol ; 54(12): 3010-3017, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27707939

RESUMEN

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.


Asunto(s)
Hibridación Genómica Comparativa/métodos , Infecciones por Haemophilus/diagnóstico , Haemophilus influenzae/clasificación , Haemophilus influenzae/genética , Lipoproteínas/genética , Fosforribosilglicinamida-Formiltransferasa/genética , Secuencia de Bases , ADN Bacteriano/genética , Genoma Bacteriano/genética , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/aislamiento & purificación , Humanos , Límite de Detección , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Análisis de Secuencia de ADN
2.
J Clin Microbiol ; 54(1): 25-34, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26311858

RESUMEN

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.


Asunto(s)
Cápsulas Bacterianas/clasificación , Portador Sano/microbiología , Técnicas de Genotipaje/métodos , Neisseria meningitidis/clasificación , Infecciones por Neisseriaceae/microbiología , Serotipificación/métodos , Adolescente , Adulto , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/inmunología , Estudios Epidemiológicos , Femenino , Humanos , Masculino , Neisseria meningitidis/genética , Neisseria meningitidis/inmunología , Adulto Joven
3.
J Infect Dis ; 211(12): 1887-94, 2015 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-25556253

RESUMEN

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.


Asunto(s)
Infecciones Meningocócicas/epidemiología , Infecciones Meningocócicas/microbiología , Vacunas Meningococicas/administración & dosificación , Neisseria meningitidis/clasificación , Neisseria meningitidis/aislamiento & purificación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Infecciones Meningocócicas/prevención & control , Persona de Mediana Edad , Neisseria meningitidis/genética , Neisseria meningitidis/inmunología , Serogrupo , Estados Unidos , Vacunas Conjugadas/administración & dosificación , Adulto Joven
4.
J Infect Dis ; 211(11): 1761-8, 2015 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-25505298

RESUMEN

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.


Asunto(s)
Portador Sano/epidemiología , Portador Sano/microbiología , Infecciones Meningocócicas/epidemiología , Infecciones Meningocócicas/microbiología , Neisseria meningitidis/inmunología , Estudiantes/estadística & datos numéricos , Adolescente , Adulto , Portador Sano/inmunología , Portador Sano/prevención & control , Femenino , Georgia/epidemiología , Humanos , Masculino , Maryland/epidemiología , Infecciones Meningocócicas/inmunología , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/inmunología , Neisseria meningitidis/clasificación , Estudios Prospectivos , Factores de Riesgo , Adulto Joven
5.
Emerg Infect Dis ; 20(1): 61-9, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24377388

RESUMEN

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.


Asunto(s)
Meningitis Bacterianas/epidemiología , Vigilancia de la Población , Niño , Preescolar , China/epidemiología , Geografía Médica , Humanos , Incidencia , Lactante , Meningitis Bacterianas/diagnóstico , Meningitis Bacterianas/microbiología , Estaciones del Año , Vigilancia de Guardia
6.
Emerg Infect Dis ; 20(3): 394-9, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24571805

RESUMEN

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.


Asunto(s)
Meningitis Meningocócica/epidemiología , Neisseria meningitidis/clasificación , Serogrupo , Adolescente , Burkina Faso/epidemiología , Niño , Preescolar , Genotipo , Historia del Siglo XXI , Humanos , Incidencia , Lactante , Recién Nacido , Meningitis Meningocócica/historia , Neisseria meningitidis/genética , Vigilancia de la Población , Adulto Joven
7.
Clin Infect Dis ; 57(3): 344-8, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23595832

RESUMEN

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.


Asunto(s)
Brotes de Enfermedades , Meningitis Meningocócica/epidemiología , Meningitis Meningocócica/microbiología , Neisseria meningitidis Serogrupo B/clasificación , Neisseria meningitidis Serogrupo B/aislamiento & purificación , Adolescente , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Factores de Riesgo , Serotipificación , Encuestas y Cuestionarios , Estados Unidos/epidemiología , Universidades , Adulto Joven
8.
BMC Infect Dis ; 13: 26, 2013 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-23339355

RESUMEN

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.


Asunto(s)
Haemophilus influenzae/genética , Meningitis Bacterianas/diagnóstico , Neisseria meningitidis/genética , Streptococcus pneumoniae/genética , Adolescente , Adulto , Anciano , Líquido Cefalorraquídeo/microbiología , Niño , Preescolar , Violeta de Genciana , Haemophilus influenzae/aislamiento & purificación , Humanos , Lactante , Recién Nacido , Persona de Mediana Edad , Neisseria meningitidis/aislamiento & purificación , Fenazinas , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Streptococcus pneumoniae/aislamiento & purificación , Adulto Joven
9.
J Bacteriol ; 194(20): 5649-56, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22904290

RESUMEN

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 ∼2-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.


Asunto(s)
Genoma Bacteriano , Neisseria meningitidis/clasificación , Neisseria meningitidis/genética , Polisacáridos Bacterianos/metabolismo , Pruebas de Aglutinación , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , Humanos , Infecciones Meningocócicas/microbiología , Datos de Secuencia Molecular , Neisseria meningitidis/inmunología , Neisseria meningitidis/aislamiento & purificación , Filogenia , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/inmunología , Análisis de Secuencia de ADN , Homología de Secuencia , Serotipificación
10.
N Engl J Med ; 360(9): 886-92, 2009 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-19246360

RESUMEN

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.


Asunto(s)
Antiinfecciosos/uso terapéutico , Ciprofloxacina/uso terapéutico , Farmacorresistencia Bacteriana/genética , Infecciones Meningocócicas/tratamiento farmacológico , Neisseria meningitidis/genética , Mutación Puntual , Adolescente , Adulto , Anciano , Secuencia de Bases , Portador Sano/microbiología , Humanos , Lactante , Infecciones Meningocócicas/microbiología , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Neisseria meningitidis/clasificación , Neisseria meningitidis/efectos de los fármacos , Neisseria meningitidis/aislamiento & purificación , Faringe/microbiología , Estados Unidos , Adulto Joven
11.
J Clin Microbiol ; 50(7): 2462-5, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22573587

RESUMEN

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.


Asunto(s)
Infecciones por Haemophilus/diagnóstico , Infecciones por Haemophilus/microbiología , Haemophilus/clasificación , Haemophilus/aislamiento & purificación , Anciano , Técnicas Bacteriológicas/métodos , Niño , ADN Bacteriano/química , ADN Bacteriano/genética , Femenino , Infecciones por Haemophilus/patología , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN , Estados Unidos , Adulto Joven
12.
J Clin Microbiol ; 50(4): 1422-4, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22301020

RESUMEN

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.


Asunto(s)
Marcadores Genéticos , Haemophilus influenzae/genética , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Inmunoglobulina D/genética , Lipoproteínas/genética , Tipificación Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Filogenia , Reacción en Cadena de la Polimerasa , ARN Bacteriano/genética , ARN Ribosómico 16S/genética
13.
J Clin Microbiol ; 50(3): 702-8, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22170919

RESUMEN

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.


Asunto(s)
Haemophilus influenzae/aislamiento & purificación , Meningitis Bacterianas/diagnóstico , Meningitis Bacterianas/microbiología , Reacción en Cadena de la Polimerasa Multiplex/métodos , Neisseria meningitidis/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Streptococcus pneumoniae/aislamiento & purificación , Haemophilus influenzae/genética , Humanos , Neisseria meningitidis/clasificación , Neisseria meningitidis/genética , Sensibilidad y Especificidad , Streptococcus pneumoniae/genética
14.
J Bacteriol ; 193(14): 3633-41, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21622743

RESUMEN

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."


Asunto(s)
Portador Sano/microbiología , Genoma Bacteriano , Infecciones Meningocócicas/microbiología , Neisseria meningitidis/genética , Polimorfismo de Nucleótido Simple , Secuencia de Bases , Variación Genética , Humanos , Datos de Secuencia Molecular , Neisseria meningitidis/clasificación , Neisseria meningitidis/aislamiento & purificación , Neisseria meningitidis/patogenicidad , Filogenia , Virulencia
15.
J Bacteriol ; 193(20): 5879-80, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21952546

RESUMEN

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.


Asunto(s)
Genoma Bacteriano , Infecciones por Haemophilus/microbiología , Haemophilus/genética , Haemophilus/aislamiento & purificación , Secuencia de Bases , Haemophilus/clasificación , Humanos , Datos de Secuencia Molecular
16.
Clin Infect Dis ; 53(12): 1230-6, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22080119

RESUMEN

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.


Asunto(s)
Infecciones por Haemophilus/epidemiología , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/clasificación , Haemophilus influenzae/aislamiento & purificación , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Etnicidad , Femenino , Infecciones por Haemophilus/mortalidad , Vacunas contra Haemophilus/administración & dosificación , Vacunas contra Haemophilus/inmunología , Humanos , Incidencia , Lactante , Masculino , Persona de Mediana Edad , Serotipificación , Estados Unidos/epidemiología , Adulto Joven
17.
J Clin Microbiol ; 49(6): 2175-82, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21471348

RESUMEN

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.


Asunto(s)
Infecciones Bacterianas/diagnóstico , Técnicas de Laboratorio Clínico/métodos , Microfluídica/métodos , Reacción en Cadena de la Polimerasa/métodos , Infecciones del Sistema Respiratorio/diagnóstico , Virosis/diagnóstico , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Humanos , Sensibilidad y Especificidad , Virus/clasificación , Virus/genética , Virus/aislamiento & purificación
18.
Bioinformatics ; 26(15): 1819-26, 2010 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-20519285

RESUMEN

MOTIVATION: New sequencing technologies have accelerated research on prokaryotic genomes and have made genome sequencing operations outside major genome sequencing centers routine. However, no off-the-shelf solution exists for the combined assembly, gene prediction, genome annotation and data presentation necessary to interpret sequencing data. The resulting requirement to invest significant resources into custom informatics support for genome sequencing projects remains a major impediment to the accessibility of high-throughput sequence data. RESULTS: We present a self-contained, automated high-throughput open source genome sequencing and computational genomics pipeline suitable for prokaryotic sequencing projects. The pipeline has been used at the Georgia Institute of Technology and the Centers for Disease Control and Prevention for the analysis of Neisseria meningitidis and Bordetella bronchiseptica genomes. The pipeline is capable of enhanced or manually assisted reference-based assembly using multiple assemblers and modes; gene predictor combining; and functional annotation of genes and gene products. Because every component of the pipeline is executed on a local machine with no need to access resources over the Internet, the pipeline is suitable for projects of a sensitive nature. Annotation of virulence-related features makes the pipeline particularly useful for projects working with pathogenic prokaryotes. AVAILABILITY AND IMPLEMENTATION: The pipeline is licensed under the open-source GNU General Public License and available at the Georgia Tech Neisseria Base (http://nbase.biology.gatech.edu/). The pipeline is implemented with a combination of Perl, Bourne Shell and MySQL and is compatible with Linux and other Unix systems.


Asunto(s)
Genoma Bacteriano/genética , Genómica/métodos , Células Procariotas , Bordetella bronchiseptica/genética , Georgia , Neisseria meningitidis/genética , Análisis de Secuencia de ADN/métodos , Programas Informáticos
19.
Int J Med Microbiol ; 301(4): 303-9, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21276750

RESUMEN

Since the implementation of Haemophilus influenzae (Hi) serotype b vaccine, other serotypes and non-typeable strains have taken on greater importance as a cause of Hi diseases. A rapid and accurate method is needed to detect all Hi regardless of the encapsulation status. We developed 2 real-time PCR (rt-PCR) assays to detect specific regions of the protein D gene (hpd). Both hpd assays are very specific and sensitive for detection of Hi. Of the 63 non-Hi isolates representing 21 bacterial species, none was detected by the hpd #1 assay, and only one of 2 H. aphrophilus isolates was detected by the hpd #3 assay. The hpd #1 and #3 assays detected 97% (229/237) and 99% (234/237) of Hi isolates, respectively, and were superior for detection of both typeable and non-typeable Hi isolates, as compared to previously developed rt-PCR targeting ompP2 or bexA. The diagnostic sensitivity and specificity of these rt-PCR assays were assessed on cerebrospinal fluid specimens collected as part of meningitis surveillance in Ulaanbaatar, Mongolia. The etiology (Neisseria meningitidis, Hi, and Streptococcus pneumoniae) of 111 suspected meningitis cases was determined by conventional methods (culture and latex agglutination), previously developed rt-PCR assays, and the new hpd assays. The rt-PCR assays were more sensitive for detection of meningitis pathogens than other classical methods and improved detection from 50% (56/111) to 75% (83/111). The hpd #3 assay identified a non-b Hi that was missed by the bexA assay and other methods. A sensitive rt-PCR assay to detect both typeable and non-typeable Hi is a useful tool for improving Hi disease surveillance especially after Hib vaccine introduction.


Asunto(s)
Bacterias/aislamiento & purificación , Técnicas Bacteriológicas/métodos , Haemophilus influenzae/aislamiento & purificación , Meningitis por Haemophilus/epidemiología , Reacción en Cadena de la Polimerasa/métodos , Bacterias/genética , Líquido Cefalorraquídeo/microbiología , Preescolar , Cartilla de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Haemophilus influenzae/genética , Humanos , Lactante , Meningitis por Haemophilus/microbiología , Datos de Secuencia Molecular , Mongolia/epidemiología , Sensibilidad y Especificidad , Análisis de Secuencia de ADN
20.
Nucleic Acids Res ; 37(Web Server issue): W606-11, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19468047

RESUMEN

The Meningococcus Genome Informatics Platform (MGIP) is a suite of computational tools for the analysis of multilocus sequence typing (MLST) data, at http://mgip.biology.gatech.edu. MLST is used to generate allelic profiles to characterize strains of Neisseria meningitidis, a major cause of bacterial meningitis worldwide. Neisseria meningitidis strains are characterized with MLST as specific sequence types (ST) and clonal complexes (CC) based on the DNA sequences at defined loci. These data are vital to molecular epidemiology studies of N. meningitidis, including outbreak investigations and population biology. MGIP analyzes DNA sequence trace files, returns individual allele calls and characterizes the STs and CCs. MGIP represents a substantial advance over existing software in several respects: (i) ease of use-MGIP is user friendly, intuitive and thoroughly documented; (ii) flexibility--because MGIP is a website, it is compatible with any computer with an internet connection, can be used from any geographic location, and there is no installation; (iii) speed--MGIP takes just over one minute to process a set of 96 trace files; and (iv) expandability--MGIP has the potential to expand to more loci than those used in MLST and even to other bacterial species.


Asunto(s)
Técnicas de Tipificación Bacteriana , Neisseria meningitidis/clasificación , Programas Informáticos , Alelos , Genoma Bacteriano , Genómica , Neisseria meningitidis/genética , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Interfaz Usuario-Computador
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