RESUMO
Real-time PCR shows the widespread presence of Coxiella burnetii DNA in a broad range of commercially available milk and milk products. MLVA genotyping shows that this is the result of the presence of a predominant C. burnetii genotype in the dairy cattle population.
Assuntos
Coxiella burnetii/classificação , Coxiella burnetii/genética , Leite/microbiologia , Tipagem Molecular , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Bovinos , Análise por Conglomerados , Coxiella burnetii/isolamento & purificação , GenótipoRESUMO
The genotypic diversity of Coxiella burnetii in clinical samples obtained from the Dutch Q fever outbreak episodes of 2007-2010 was determined by using a 6-locus variable-number tandem repeat analysis panel. The results are consistent with the introduction of one founder genotype that is gradually diversifying over time while spreading throughout The Netherlands.
Assuntos
Coxiella burnetii/classificação , Coxiella burnetii/genética , Surtos de Doenças , Variação Genética , Febre Q/epidemiologia , Febre Q/microbiologia , Coxiella burnetii/isolamento & purificação , Genótipo , Humanos , Repetições Minissatélites , Epidemiologia Molecular , Tipagem Molecular , Países Baixos/epidemiologiaRESUMO
By performing genotyping, a laboratory contamination involving Q fever was traced back to the antigen preparation used in a commercially available complement fixation test. It was established that such antigen preparations contain relatively high loads of DNA/RNA, making them potential sources of contamination but also convenient preparations for control material.
Assuntos
Testes de Fixação de Complemento , Coxiella burnetii/classificação , Coxiella burnetii/genética , Contaminação por DNA , Kit de Reagentes para Diagnóstico/microbiologia , HumanosRESUMO
Contamination of an in-house diagnostic real-time PCR for Q fever was traced back to a commercially obtained PCR Master Mix. It was established that this Master Mix contained DNA from Coxiella burnetii, probably as a result of the use of compounds of animal origin such as bovine serum albumin.
Assuntos
Soluções Tampão , Coxiella burnetii/genética , DNA/análise , Reação em Cadeia da Polimerase , Kit de Reagentes para Diagnóstico , DNA/genética , HumanosRESUMO
In the Netherlands, there is an ongoing and unparalleled outbreak of Q fever. Rapid and reliable methods to identify patients infected with Coxiella burnetii, the causative agent of Q fever, are urgently needed. We evaluated the performance of different DNA extraction methods and real-time PCR assays that are in use in seven diagnostic or reference laboratories in the Netherlands. A low degree of variation in the sensitivities of most of the developed real-time PCR assays was observed. However, PCR assays amplifying short DNA fragments yielded better results than those producing large DNA fragments. With regard to DNA extraction, the automated MagNA Pure Compact system and the manual QIAamp DNA mini kit consistently yielded better results than either the MagNA Pure LC system and NucliSens EasyMag (both automated) or the High Pure viral nucleic acid kit (manual). The present study shows that multiple combinations of DNA extraction kits and real-time PCR assays offer equivalent solutions to detect C. burnetii DNA in serum samples from patients suspected to have Q fever.
Assuntos
Técnicas Bacteriológicas/métodos , Coxiella burnetii/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Febre Q/diagnóstico , Soro/microbiologia , Coxiella burnetii/genética , Humanos , Países Baixos , Febre Q/microbiologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Broad-spectrum analysis for pathogens in patients with respiratory tract infections is becoming more relevant as the number of potential infectious agents is still increasing. Here we describe the new multiparameter RespiFinder assay, which is based on the multiplex ligation-dependent probe amplification (MLPA) technology. This assay detects 15 respiratory viruses in one reaction. The MLPA reaction is preceded by a preamplification step which ensures the detection of both RNA and DNA viruses with the same specificity and sensitivity as individual monoplex real-time reverse transcription-PCRs. The RespiFinder assay was validated with 144 clinical samples, and the results of the assay were compared to those of cell culture and a respiratory syncytial virus (RSV)-specific immunochromatography assay (ICA). Compared to the cell culture results, the RespiFinder assay showed specificities and sensitivities of 98.2% and 100%, respectively, for adenovirus; 96.4% and 100%, respectively, for human metapneumovirus; 98.2% and 100%, respectively, for influenza A virus (InfA); 99.1% and 100%, respectively, for parainfluenza virus type 1 (PIV-1); 99.1% and 80%, respectively, for PIV-3; 90.1% and 100%, respectively, for rhinovirus; and 94.6% and 100%, respectively, for RSV. Compared to the results of the RSV-specific ICA, the RespiFinder assay gave a specificity and a sensitivity of 82.4% and 80%, respectively. PIV-2, PIV-4, influenza B virus, InfA H5N1, and coronavirus 229E were not detected in the clinical specimens tested. The use of the RespiFinder assay resulted in an increase in the diagnostic yield compared to that obtained by cell culture (diagnostic yields, 60% and 35.5%, respectively). In conclusion, the RespiFinder assay provides a user-friendly and high-throughput tool for the simultaneous detection of 15 respiratory viruses with excellent overall performance statistics.
Assuntos
Vírus de DNA/isolamento & purificação , Vírus de RNA/isolamento & purificação , Doenças Respiratórias/virologia , Viroses/virologia , Adulto , Criança , Pré-Escolar , Primers do DNA , Vírus de DNA/classificação , Vírus de DNA/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico , Infecções por Vírus de RNA/virologia , Vírus de RNA/classificação , Vírus de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Cultura de VírusAssuntos
Coxiella burnetii/genética , Genótipo , Febre Q/epidemiologia , Animais , Doenças das Cabras/epidemiologia , Doenças das Cabras/microbiologia , Cabras , Humanos , Tipagem de Sequências Multilocus , Países Baixos/epidemiologia , Febre Q/microbiologia , Febre Q/veterinária , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/microbiologiaAssuntos
Doenças Transmissíveis Emergentes/epidemiologia , Surtos de Doenças , Febre Q/epidemiologia , Animais , Sequência de Bases , Doenças Transmissíveis Emergentes/microbiologia , Doenças Transmissíveis Emergentes/veterinária , Coxiella burnetii/genética , Coxiella burnetii/isolamento & purificação , Coxiella burnetii/patogenicidade , DNA Bacteriano/genética , Surtos de Doenças/veterinária , Feminino , Genótipo , Cabras , Humanos , Masculino , Países Baixos/epidemiologia , Gravidez , Febre Q/microbiologia , Febre Q/veterinária , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/microbiologiaRESUMO
In the Netherlands an increasing number of laboratories are involved in diagnosing acute Q-fever. More uniformity in diagnostics and interpretation is desirable. To enable this, a working group on diagnostics of acute Q-fever was created on the initiative of the National Institute for Public Health and the Environment (RIVM) and the Dutch Association for Medical Microbiology (NVMM). The diagnostics of acute Q-fever includes a diagnostic flow chart (algorithm) consisting of tests for DNA and for antibodies against the antigens that appear in the successive stages of the disease. Reporting of both confirmed and suspected cases of acute Q-fever is obligatory.
Assuntos
Anticorpos Antibacterianos/sangue , Coxiella burnetii/isolamento & purificação , DNA Bacteriano/sangue , Laboratórios/normas , Febre Q/diagnóstico , Doença Aguda , Diagnóstico Precoce , Humanos , Países BaixosRESUMO
A new human papillomavirus (HPV) assay using high-density DNA microarrays is described. An HPV DNA fragment from the 3' end of the E1 gene was amplified and digoxigenin labeled by PCR, and the resulting amplicons were hybridized onto type-specific oligonucleotides immobilized on high-density DNA microarrays. For detection, a simple immunohistochemical staining procedure was used with a substrate that has both colorimetric and fluorescent properties. This detection chemistry enables the rapid identification of reactive spots by regular light microscopy and semiquantification by laser scanning. Both single and multiple HPV infections are recognized by this assay, and the corresponding HPV types are easily identified. With this assay, 53 mucosal HPV types were detected and identified. A total of 45 HPV types were identified by a single type-specific probe, whereas the remaining 8 mucosal HPV types could be identified by a specific combination of probes. The simple assay format allows usage of this assay without expensive equipment, making it accessible to all diagnostic laboratories with PCR facilities.