RESUMO
Whole-genome sequencing (WGS) of microbial pathogens recovered from patients with infectious disease facilitates high-resolution strain characterization and molecular epidemiology. However, increasing reliance on culture-independent methods to diagnose infectious diseases has resulted in few isolates available for WGS. Here, we report a novel culture-independent approach to genome characterization of Bordetella pertussis, the causative agent of pertussis and a paradigm for insufficient genomic surveillance due to limited culture of clinical isolates. Sequencing libraries constructed directly from residual pertussis-positive diagnostic nasopharyngeal specimens were hybridized with biotinylated RNA "baits" targeting B. pertussis fragments within complex mixtures that contained high concentrations of host and microbial background DNA. Recovery of B. pertussis genome sequence data was evaluated with mock and pooled negative clinical specimens spiked with reducing concentrations of either purified DNA or inactivated cells. Targeted enrichment increased the yield of B. pertussis sequencing reads up to 90% while simultaneously decreasing host reads to less than 10%. Filtered sequencing reads provided sufficient genome coverage to perform characterization via whole-genome single nucleotide polymorphisms and whole-genome multilocus sequencing typing. Moreover, these data were concordant with sequenced isolates recovered from the same specimens such that phylogenetic reconstructions from either consistently clustered the same putatively linked cases. The optimized protocol is suitable for nasopharyngeal specimens with diagnostic IS481 Ct < 35 and >10 ng DNA. Routine implementation of these methods could strengthen surveillance and study of pertussis resurgence by capturing additional cases with genomic characterization.
Assuntos
Bordetella , Coqueluche , Humanos , Bordetella pertussis/genética , Coqueluche/diagnóstico , Coqueluche/epidemiologia , Filogenia , Genômica , DNARESUMO
BACKGROUND: Diphtheria, a life-threatening respiratory disease, is caused mainly by toxin-producing strains of Corynebacterium diphtheriae, while nontoxigenic corynebacteria (eg, Corynebacterium pseudodiphtheriticum) rarely causes diphtheria-like illness. Recently, global diphtheria outbreaks have resulted from breakdown of health care infrastructures, particularly in countries experiencing political conflict. This report summarizes a laboratory and epidemiological investigation of a diphtheria outbreak among forcibly displaced Myanmar nationals in Bangladesh. METHODS: Specimens and clinical information were collected from patients presenting at diphtheria treatment centers. Swabs were tested for toxin gene (tox)-bearing C. diphtheriae by real-time polymerase chain reaction (RT-PCR) and culture. The isolation of another Corynebacterium species prompted further laboratory investigation. RESULTS: Among 382 patients, 153 (40%) tested tox positive for C. diphtheriae by RT-PCR; 31 (20%) PCR-positive swabs were culture confirmed. RT-PCR revealed 78% (298/382) of patients tested positive for C. pseudodiphtheriticum. Of patients positive for only C. diphtheriae, 63% (17/27) had severe disease compared to 55% (69/126) positive for both Corynebacterium species, and 38% (66/172) for only C. pseudodiphtheriticum. CONCLUSIONS: We report confirmation of a diphtheria outbreak and identification of a cocirculating Corynebacterium species. The high proportion of C. pseudodiphtheriticum codetection may explain why many suspected patients testing negative for C. diphtheriae presented with diphtheria-like symptoms.
Assuntos
Corynebacterium diphtheriae , Corynebacterium , Difteria , Corynebacterium/isolamento & purificação , Corynebacterium diphtheriae/isolamento & purificação , Difteria/epidemiologia , Toxina Diftérica , Surtos de Doenças , Humanos , Mianmar/epidemiologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
We characterized 170 complete genome assemblies from clinical Bordetella pertussis isolates representing geographic and temporal diversity in the United States. These data capture genotypic shifts, including increased pertactin deficiency, occurring amid the current pertussis disease resurgence and provide a foundation for needed research to direct future public health control strategies.
Assuntos
Bordetella pertussis/classificação , Bordetella pertussis/genética , Variação Genética , Genoma Bacteriano , Genômica , Coqueluche/epidemiologia , Coqueluche/microbiologia , Genômica/métodos , Geografia Médica , História do Século XXI , Humanos , Filogenia , Polimorfismo de Nucleotídeo Único , Vigilância em Saúde Pública , Estados Unidos/epidemiologia , Coqueluche/históriaRESUMO
BACKGROUND: Pertussis remains a cause of morbidity and mortality among young infants. There are limited data on the pertussis disease burden in this age group from low- and lower-middle-income countries, including in South Asia. METHODS: We conducted an active community-based surveillance study from February 2015 to April 2016 among 2 cohorts of young infants in 4 low-income settlements in Karachi, Pakistan. Infants were enrolled either at birth (closed cohort) or at ages up to 10 weeks (open cohort) and followed until 18 weeks of age. Nasopharyngeal swab specimens were obtained from infants who met a standardized syndromic case definition and tested for Bordetella pertussis using real-time polymerase chain reaction. We determined the incidence of pertussis using a protocol-defined case definition, as well as the US Centers for Disease Control and Prevention (CDC) definitions for confirmed and probable pertussis. RESULTS: Of 2021 infants enrolled into the study, 8 infants met the protocol-defined pertussis case definition, for an incidence of 3.96 (95% confidence interval [CI], 1.84-7.50) cases per 1000 infants. Seven of the pertussis cases met the CDC pertussis case definition (5 confirmed, 2 probable), for incidences of CDC-defined confirmed pertussis of 2.47 (95% CI, .90-5.48) cases per 1000 infants, and probable pertussis of 0.99 (95% CI, .17-3.27) cases per 1000 infants. Three of the pertussis cases were severe according to the Modified Preziosi Scale score. CONCLUSIONS: In one of the first prospective surveillance studies of infant pertussis in a developing country, we identified a moderate burden of pertussis disease in early infancy in Pakistan.
Assuntos
Coqueluche/epidemiologia , Bordetella pertussis , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Paquistão/epidemiologia , Vigilância da População , Estudos Prospectivos , Estações do Ano , Índice de Gravidade de Doença , Fatores Socioeconômicos , Coqueluche/diagnósticoRESUMO
BACKGROUND: A recent increase in Bordetella pertussis without the pertactin protein, an acellular vaccine immunogen, has been reported in the United States. Determining whether pertactin-deficient (PRN(-)) B. pertussis is evading vaccine-induced immunity or altering the severity of illness is needed. METHODS: We retrospectively assessed for associations between pertactin production and both clinical presentation and vaccine history. Cases with isolates collected between May 2011 and February 2013 from 8 states were included. We calculated unadjusted and adjusted odds ratios (ORs) using multivariable logistic regression analysis. RESULTS: Among 753 isolates, 640 (85%) were PRN(-). The age distribution differed between cases caused by PRN(-) B. pertussis and cases caused by B. pertussis producing pertactin (PRN(+)) (P = .01). The proportion reporting individual pertussis symptoms was similar between the 2 groups, except a higher proportion of PRN(+) case-patients reported apnea (P = .005). Twenty-two case-patients were hospitalized; 6% in the PRN(+) group compared to 3% in the PRN(-) group (P = .11). Case-patients having received at least 1 pertussis vaccine dose had a higher odds of having PRN(-) B. pertussis compared with unvaccinated case-patients (adjusted OR = 2.2; 95% confidence interval [CI], 1.3-4.0). When restricted to case-patients at least 1 year of age and those age-appropriately vaccinated, the adjusted OR increased to 2.7 (95% CI, 1.2-6.1). CONCLUSIONS: The significant association between vaccination and isolate pertactin production suggests that the likelihood of having reported disease caused by PRN(-) compared with PRN(+) strains is greater in vaccinated persons. Additional studies are needed to assess whether vaccine effectiveness is diminished against PRN(-) strains.
Assuntos
Proteínas da Membrana Bacteriana Externa/análise , Proteínas da Membrana Bacteriana Externa/genética , Bordetella pertussis/genética , Bordetella pertussis/isolamento & purificação , Vacina contra Coqueluche/administração & dosagem , Fatores de Virulência de Bordetella/análise , Fatores de Virulência de Bordetella/genética , Coqueluche/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas da Membrana Bacteriana Externa/imunologia , Western Blotting , Bordetella pertussis/imunologia , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Evasão da Resposta Imune , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Estados Unidos/epidemiologia , Fatores de Virulência de Bordetella/imunologia , Coqueluche/imunologia , Coqueluche/patologia , Adulto JovemRESUMO
In the United States, the general laboratory method for diagnosing pertussis, caused by Bordetella pertussis, is real-time PCR (rt-PCR) targeting insertion sequence 481 (IS481). Other Bordetella species (parapertussis, holmesii, and bronchiseptica) can also cause a pertussis-like syndrome, and some commercial laboratory assays include the insertion sequence 1001 (pIS1001) that can detect B. parapertussis/B. bronchiseptica (BppBb). Because IS481 exists in B. pertussis and B. holmesii, current commercial assays cannot differentiate these two species. We used a multiplex rt-PCR assay containing species-specific targets to Bordetella to evaluate clinical specimens detected as B. pertussis/B. holmesii (BpBh) or BppBb by commercial laboratories. A sample of 3,984 clinical specimens positive for IS481 or pIS1001 from two commercial laboratories during 2012-2019 were re-tested at CDC. Agreement of Bordetella species between the CDC and commercial laboratory assays, and the proportion of commercial laboratory specimens that were non-B. pertussis by CDC's assay was assessed. Overall agreement in Bordetella species detection and identification between the CDC and commercial lab assays was 85%. Agreement for identifying B. pertussis was 87% for 3,663 BpBh specimens and 98% for identifying B. parapertussis in 310 BppBb specimens. CDC's assay detected B. holmesii in 55/3,984 (1.4%) specimens. Most discrepant results (410/490, 82%) were BpBh specimens interpreted as indeterminate B. pertussis at CDC. We found a small portion of B. holmesii in a sample of IS481-positive clinical specimens originally identified by commercial laboratory rt-PCR assays, suggesting that commercial PCR assays are a reliable diagnostic tool for correctly identifying Bordetella species in most patients with suspected pertussis. IMPORTANCE: When testing specimens collected from patients with suspected pertussis, large-scale commercial laboratories in the United States employ an IS481-based assay that cannot differentiate between Bordetella pertussis and Bordetella holmseii. The level of B. holmesii causing pertussis-like illness in the United States is not well-understood given that only B. pertussis is nationally notifiable. After re-testing with a multiplex, species-specific rt-PCR assay, our data show low levels of B. holmesii identified in a sample of IS481-positive clinical specimens originally identified by commercial laboratory rt-PCR assays. These results reinforce the validity of large-scale commercial rt-PCR testing as a reliable diagnostic tool for pertussis in the United States.
Assuntos
Infecções por Bordetella , Bordetella pertussis , Bordetella , Reação em Cadeia da Polimerase em Tempo Real , Estados Unidos , Humanos , Bordetella/genética , Bordetella/classificação , Bordetella/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções por Bordetella/microbiologia , Infecções por Bordetella/diagnóstico , Bordetella pertussis/genética , Bordetella pertussis/classificação , Bordetella pertussis/isolamento & purificação , Coqueluche/diagnóstico , Coqueluche/microbiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Sensibilidade e Especificidade , Elementos de DNA Transponíveis/genética , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/isolamento & purificação , Bordetella bronchiseptica/classificaçãoRESUMO
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.
Assuntos
Genoma Bacteriano/genética , Genômica/métodos , Células Procarióticas , Bordetella bronchiseptica/genética , Georgia , Neisseria meningitidis/genética , Análise de Sequência de DNA/métodos , SoftwareRESUMO
Novel nontoxigenic Corynebacterium diphtheriae was isolated from a domestic cat with severe otitis. Contact investigation and carrier study of human and animal contacts yielded 3 additional, identical isolates from cats, although no evidence of zoonotic transmission was identified. Molecular methods distinguished the feline isolates from known C. diphtheriae.
Assuntos
Doenças do Gato/microbiologia , Corynebacterium diphtheriae/isolamento & purificação , Difteria/veterinária , Animais , Animais Domésticos/microbiologia , Gatos/microbiologia , Corynebacterium diphtheriae/genética , Difteria/microbiologia , Feminino , Humanos , Filogenia , West VirginiaRESUMO
Bordetella pertussis (Bp) is the causative agent of pertussis, a vaccine preventable disease occurring primarily in children. In recent years, there has been increased reporting of pertussis. Current pertussis vaccines are acellular and consist of Bp proteins including the major virulence factor pertussis toxin (Ptx), a 5-subunit exotoxin. Variation in Ptx subunit amino acid (AA) sequence could possibly affect the immune response. A blind comparative mass spectrometric (MS) analysis of commercially available Ptx as well as the chemically modified toxoid (Ptxd) from licensed vaccines was performed to assess peptide sequence and AA coverage variability as well as relative amounts of Ptx subunits. Qualitatively, there are similarities among the various sources based on AA percent coverages and MS/MS fragmentation profiles. Additionally, based on a label-free mass spectrometry-based quantification method there is differential relative abundance of the subunits among the sources.
RESUMO
The ResPlex I assay (Qiagen) was designed to amplify and detect DNA of six bacterial respiratory pathogens. This assay was compared with real-time PCR assays based upon the same target sequences for the ability detect the target bacteria by use of both stock strains and specimens from respiratory disease patients. The ResPlex I assay is somewhat less sensitive than real-time PCR assays but offers the advantage of multiple assays in a single reaction.
Assuntos
Bactérias/classificação , Reação em Cadeia da Polimerase/métodos , Kit de Reagentes para Diagnóstico , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/microbiologia , Bactérias/genética , Bactérias/isolamento & purificação , Infecções Bacterianas/microbiologia , Primers do DNA , DNA Bacteriano/análise , Humanos , Sistema Respiratório/microbiologia , Sensibilidade e EspecificidadeRESUMO
Streptococcus pneumoniae is the most frequent cause of pneumonia and meningitis. Because S pneumoniae can colonize the upper respiratory tract and antibiotic treatment may inhibit growth, culture-based diagnosis can be problematic. An immunohistochemical assay using a polyclonal antibody against pneumococci was used to test formalin-fixed, paraffin-embedded tissue samples from 46 patients for whom bacterial culture results were available. Samples from 26 patients demonstrated pneumococcal antigens in areas of pneumonia, meningitis, or osteomyelitis or within circulating inflammatory cells. Various specimens from 18 patients grew S pneumoniae, whereas 8 had cultures that grew mixed bacteria or had no growth but were polymerase chain reaction-positive for the S pneumoniae Ity A gene. Pneumococcal antigens were not present in 20 cases (7 grew Streptococcus pyogenes; 9, Staphylococcus aureus; and 4, Haemophilus influenzae). Compared with culture, the immunohistochemical assay showed 100% sensitivity and 71% specificity. Immunohistochemical analysis has the diagnostic advantage of correlating host inflammatory reaction with presence of pneumococci.
Assuntos
Imuno-Histoquímica , Infecções Pneumocócicas/diagnóstico , Coloração e Rotulagem , Streptococcus pneumoniae/isolamento & purificação , Adulto , Idoso , Técnicas de Cultura de Células , Feminino , Formaldeído , Violeta Genciana , Humanos , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Fenazinas , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos , Fixação de TecidosRESUMO
OBJECTIVES: To characterize illness and identify the etiology for two nursing home outbreaks of respiratory illness. DESIGN: Multisite outbreak investigations; cohort. SETTING: Two nursing homes in Pennsylvania. PARTICIPANTS: Facility A residents (n = 170), Facility B residents (n = 124), and employees (n = 91). MEASUREMENTS: Medical records for Facility A and B residents were reviewed, and employees from Facility B self-administered a questionnaire to identify risk factors for illness. Serological, oropharyngeal, and nasopharyngeal specimens were collected for both outbreaks, and testing for respiratory pathogens was performed. RESULTS: In Facility A, 40 (24%) of 170 residents were identified with respiratory illness; 13 (33%) case-patients had radiographically confirmed pneumonia, 15 (38%) were taken to a hospital, and two (5%) died. Of 10 specimens collected from symptomatic Facility A case-patients, four (40%) tested positive using reverse transcription polymerase chain reaction for rhinovirus. In Facility B, 77 (62%) of 124 residents had respiratory illness, and 40 (52%) had radiographically confirmed pneumonia; 12 (16%) case-patients were hospitalized, and five (6%) died. Of 19 respiratory specimens collected from symptomatic Facility B case-patients, six (32%) were positive for rhinovirus; one was from an employee. Five (50%) of 10 rhinovirus-positive cases in both outbreaks had clinical and radiographic evidence of pneumonia. CONCLUSION: These investigations suggest that rhinoviruses may be an underrecognized cause of respiratory outbreaks in nursing homes, capable of causing pneumonia and perhaps death.
Assuntos
Surtos de Doenças , Casas de Saúde , Infecções por Picornaviridae/epidemiologia , Infecções Respiratórias/epidemiologia , Rhinovirus , Doença Aguda , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/imunologia , DNA Viral/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pennsylvania/epidemiologia , Infecções por Picornaviridae/virologia , Infecções Respiratórias/virologia , Rhinovirus/genética , Rhinovirus/imunologiaRESUMO
An increasing number of pertussis-like cases are attributed to the emergent pathogen Bordetella holmesii. The genomes of 9 clinical isolates show that they are clonal, lack the virulence factors encoded by B. pertussis, and are more similar to nonpertussis bordetellae. New markers for B. holmesii can be developed using these sequences.
RESUMO
Bacteria of the genus Corynebacterium are important primary and opportunistic pathogens. Many are zoonotic agents. In this report, phenotypic (API Coryne analysis), genetic (rpoB and 16S rRNA gene sequencing), and physical methods (MS) were used to distinguish the closely related diphtheroid species Corynebacterium ulcerans and Corynebacterium pseudotuberculosis, and to definitively diagnose Corynebacterium renale from cephalic implants of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques used in cognitive neuroscience research. Throat and cephalic implant cultures yielded 85 isolates from 43 macaques. Identification by API Coryne yielded C. ulcerans (nâ=â74), Corynebacterium pseudotuberculosis (nâ=â2), C. renale or most closely related to C. renale (nâ=â3), and commensals and opportunists (nâ=â6). The two isolates identified as C. pseudotuberculosis by API Coryne required genetic and MS analysis for accurate characterization as C. ulcerans. Of three isolates identified as C. renale by 16S rRNA gene sequencing, only one could be confirmed as such by API Coryne, rpoB gene sequencing and MS. This study emphasizes the importance of adjunct methods in identification of coryneforms and is the first isolation of C. renale from cephalic implants in macaques.
Assuntos
Infecções por Corynebacterium/veterinária , Corynebacterium/classificação , Corynebacterium/isolamento & purificação , Macaca , Doenças dos Macacos/microbiologia , Infecções Relacionadas à Prótese/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Corynebacterium/genética , Infecções por Corynebacterium/microbiologia , DNA Bacteriano/química , DNA Ribossômico/química , Feminino , Regulação Bacteriana da Expressão Gênica/fisiologia , Masculino , Orofaringe/microbiologia , Filogenia , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In an effort to improve the reliability and reproducibility of serological assays for Bordetella pertussis, a collaborative study was conducted to compare four different sources of pertussis toxin (PT) as coating antigens in the immunoglobulin G (IgG) anti-PT enzyme-linked immunosorbent assay (ELISA). Four sources of PT were used as coating antigens in the IgG anti-PT ELISA in four different testing laboratories (labs A to D) to determine whether the different antigen preparations and different laboratories influenced assay results. A panel of 60 sera consisting of deidentified human specimens from previous vaccination trials of healthy adults and infants and clinical specimens from outbreak settings was tested. In the four laboratories, each sample was tested three times with the four PT antigens according to the standard coating optimization and IgG anti-PT ELISA testing procedures used in that laboratory. Differences among the antigens, as well as intra- and interlaboratory variability, were evaluated. Excellent agreement was observed with the test panel results among the four antigens within each laboratory. Concordance correlation coefficient (r(c)) measurements among the different antigens ranged from 0.99, 0.99 to 1.00, 1.00, and 0.97 to 1.00 for labs A to D, respectively. The comparisons between pairs of laboratories also indicated a high degree of concordance for each PT preparation, with r(c) measurements between 0.90 and 0.98, 0.93 and 0.99, 0.92 and 0.98, and 0.93 and 0.99 for antigens 1 to 4, respectively. Relatively minor differences in results were observed among laboratories or among antigens, suggesting that the four PT antigens are quite similar and could be considered for acceptance in harmonized immunoassays used for serodiagnosis or vaccine evaluation.
Assuntos
Anticorpos Antibacterianos/sangue , Bordetella pertussis/imunologia , Técnicas de Laboratório Clínico/métodos , Imunoglobulina G/sangue , Toxina Pertussis , Coqueluche/diagnóstico , Coqueluche/imunologia , Adulto , Pré-Escolar , Técnicas de Laboratório Clínico/normas , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Lactente , Reprodutibilidade dos TestesRESUMO
Bordetella pertussis (Bp) is the etiologic agent of pertussis or whooping cough, a highly contagious respiratory disease occurring primarily in infants and young children. Although vaccine preventable, pertussis cases have increased over the years leading researchers to re-evaluate vaccine control strategies. Since bacterial outer membrane proteins, comprising the surfaceome, often play roles in pathogenesis and antibody-mediated immunity, three recent Bp circulating isolates were examined using proteomics to identify any potential changes in surface protein expression. Fractions enriched for outer membrane proteins were digested with trypsin and the peptides analyzed by nano liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS), followed by database analysis to elucidate the surfaceomes of our three Bp isolates. Furthermore, a less labor intensive non-gel based antibody affinity capture technology in conjunction with MS was employed to assess each Bp strains' immunogenic outer membrane proteins. This novel technique is generally applicable allowing for the identification of immunogenic surface expressed proteins on pertussis and other pathogenic bacteria.
Assuntos
Proteínas de Bactérias/análise , Bordetella pertussis/química , Proteoma/análise , Proteômica/métodos , Coqueluche/microbiologia , Animais , Anticorpos Antibacterianos/metabolismo , Antígenos de Bactérias/isolamento & purificação , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/isolamento & purificação , Bordetella pertussis/isolamento & purificação , Pré-Escolar , Cromatografia Líquida/métodos , Biologia Computacional/métodos , Feminino , Humanos , Lactente , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Proteoma/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Tetramethylrhodamine (TAMRA) and black hole quencher 1 (BHQ1) quenched probes and five one-step RT-PCR kits were evaluated in TaqMan real-time RT-PCR assays for detection of respiratory pathogens. The intra-assay variability of the BHQ1 probes were 1.2-2.8-fold lower than those of the TAMRA probes. All kits amplified the specific targets, but differed in their sensitivity by up to 3 orders of magnitude. The AgPath-ID kit provided the best overall performance for all assay targets.
Assuntos
Sondas de DNA , Corantes Fluorescentes , Kit de Reagentes para Diagnóstico , Sistema Respiratório , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rodaminas , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Infecções Bacterianas/virologia , Corantes Fluorescentes/classificação , Corantes Fluorescentes/metabolismo , Humanos , Sistema Respiratório/microbiologia , Sistema Respiratório/virologia , Rodaminas/metabolismo , Taq Polimerase , Viroses/virologia , Vírus/classificação , Vírus/genética , Vírus/isolamento & purificaçãoRESUMO
Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute respiratory diseases such as pneumonia and bronchitis. Previous studies have established that C. pneumoniae can induce cytokines in mouse and/or human cells, but little information is available on the cytokine response of respiratory epithelial cells, a first line of infection. In this study, heparin treatment of C. pneumoniae significantly reduced its ability to induce interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) mRNA in human lung carcinoma cells, indicating that cytadherence is an important early stimulus for induction of proinflammatory mediators. Although the IL-8, gamma interferon, and TNF-alpha message was consistently induced by infection of A549 cells not treated with heparin, only an elevation of IL-8 protein was detected in A549 supernatants. A549 IL-beta and IL-6 mRNA and supernatant protein profiles were not significantly changed by infection. Heat or UV inactivation of C. pneumoniae only partially reduced the cytokine response, and inhibition of C. pneumoniae protein or DNA synthesis did not affect its ability to induce cytokine gene expression. To prevent stress-induced cytokine release by the A549 cells, centrifugation was not utilized for infection experiments. These experiments establish the importance of cytadherence in cytokine release by cells of respiratory epithelial origin and suggest that further work in the area of cytokine mediators is warranted to gain valuable pathogenic and therapeutic insights.
Assuntos
Chlamydophila pneumoniae/imunologia , Chlamydophila pneumoniae/patogenicidade , Citocinas/biossíntese , Células Epiteliais/microbiologia , Inflamação/imunologia , Pulmão/microbiologia , Aderência Bacteriana , Citocinas/genética , Temperatura Alta , Humanos , Interleucina-8/biossíntese , Interleucina-8/genética , Pulmão/citologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Células Tumorais CultivadasRESUMO
In response to the emergence of severe acute respiratory syndrome (SARS), the United States established national surveillance using a sensitive case definition incorporating clinical, epidemiologic, and laboratory criteria. Of 1,460 unexplained respiratory illnesses reported by state and local health departments to the Centers for Disease Control and Prevention from March 17 to July 30, 2003, a total of 398 (27%) met clinical and epidemiologic SARS case criteria. Of these, 72 (18%) were probable cases with radiographic evidence of pneumonia. Eight (2%) were laboratory-confirmed SARS-coronavirus (SARS-CoV) infections, 206 (52%) were SARS-CoV negative, and 184 (46%) had undetermined SARS-CoV status because of missing convalescent-phase serum specimens. Thirty-one percent (124/398) of case-patients were hospitalized; none died. Travel was the most common epidemiologic link (329/398, 83%), and mainland China was the affected area most commonly visited. One case of possible household transmission was reported, and no laboratory-confirmed infections occurred among healthcare workers. Successes and limitations of this emergency surveillance can guide preparations for future outbreaks of SARS or respiratory diseases of unknown etiology.