RESUMEN
BACKGROUND: In Germany, the outbreak of the novel pandemic 2009 influenza A(H1N1) virus A(H1N1)pdm09 caused a wave of high activity between November 2009 and January 2011. The aim of this study was to investigate the prevalence of 19 respiratory pathogens in children hospitalized for lower respiratory tract infections during the winter influenza seasons of 2009/2010 and 2010/2011 and to observe a possible impact of influenza A(H1N1)pdm09 on the epidemiology of other epidemic viruses. MATERIALS AND METHODS: Specimens were nasopharyngeal aspirates which had been collected from children admitted to the participating hospitals in the area of Mainz, Wiesbaden, and Kiel, Germany, with acute community-acquired lower respiratory tract infections. The specimens were subjected to a previously described multiplex reverse transcription PCR assay to detect the following microorganisms: enterovirus, influenza virus types A and B, respiratory syncytial virus (RSV), parainfluenzavirus types 1-4, adenovirus, Mycoplasma pneumoniae, Chlamydophila pneumoniae, rhinovirus, human metapneumovirus (hMPV), coronavirus OC43 and 229E, influenza A(H1N1)pdm09, Bordetella pertussis, Bordetella parapertussis, and Legionella pneumophila. RESULTS: A total of 3,998 clinical specimens were collected from July 2009 to March 2011, of which 296 were positive for A(H1N1)pdm09. An epidemic of seasonal influenza A or B was not observed in the 2009/2010 season, but a minor epidemic of seasonal influenza B was observed in January/February 2011. Influenza A(H1N1)pdm09 coincided with the absence of the seasonal influenza A of former years. The RSV and hMPV epidemics of 2009/2010 erupted several weeks later than expected based on data collected in the PID-ARI-Network during the past 10 years, whereas in the 2010/2011 influenza season they occurred as expected. CONCLUSIONS: The emergence of the novel influenza A(H1N1)pdm09 virus may have been influenced the epidemiology of other epidemic viruses, such as the RSV and hMPV. No epidemic of seasonal influenza was observed in the 2009/2010 influenza season.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/epidemiología , Pandemias , Infecciones del Sistema Respiratorio/epidemiología , Enfermedad Aguda/epidemiología , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Alemania/epidemiología , Hospitalización , Humanos , Lactante , Gripe Humana/virología , Reacción en Cadena de la Polimerasa Multiplex , Prevalencia , Infecciones del Sistema Respiratorio/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estaciones del Año , Virus/aislamiento & purificaciónRESUMEN
INTRODUCTION: Since acute respiratory tract infections inflict a high burden of disease in children worldwide, a multiplex reverse transcription polymerase chain reaction combined with a microwell hybridization assay (m-RT-PCR-ELISA) to detect 19 different respiratory pathogens was developed and validated. METHODS: A total of 430 respiratory specimens were retrospectively tested in parallel by both the advanced 19-valent m-RT-PCR-ELISA as well as by culture or individual RT-PCR assays used in clinical routine. RESULTS: The mean (median) sensitivity of the m-RT-PCR-ELISA in the retrospective test was 93.3% (95.1%; range 83.3-100 %), and the mean (median) specificity was 99.8 and 100 % (range 98.6-100 %), respectively. The mean positive predictive value was 99.3 % (range 93.4-100 %) and the mean negative predictive value was 95.3 % (range 98.4-100 %). Feasibility and clinical value of the 19-valent method was prospectively shown on 16,231 incoming clinical specimens from patients between 0 and 16 years of age with acute respiratory tract infections admitted to pediatric hospitals or private practices from October 2003 to June 2010 in three regions in Germany (Kiel, Mainz, Freiburg; Freiburg to June 2007 only). At least one microorganism was detected in 10,765 of 16,231 (66.3 %) clinical specimens: 5,044 RV, 1,999 RSV, 1,286 AV, 944 EV, 737 seasonal IVA, 173 pandemic IVA H1N1-2009, 899 MPV, 518 CV, 383 PIV3, 268 PIV1, 259 Mpn, 205 IVB, 164 PIV2, 144 PIV4, 103 Bp, 29 Cpn and 29 Bpp, while reovirus and Lpn were not present in these specimens from a pediatric population. More than one organism could be detected in 13.4 % of the specimens. CONCLUSIONS: The m-RT-PCR-ELISA evaluated here improves the spectrum for diagnosing respiratory infections and is a feasible instrument for individual diagnostic and epidemiological studies.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática , Reacción en Cadena de la Polimerasa Multiplex , Infecciones del Sistema Respiratorio/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Humanos , Vigilancia de la Población , Reproducibilidad de los Resultados , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , Sensibilidad y EspecificidadRESUMEN
The Directigen Flu A+B enzyme immunoassay and the Abbott TestPack RSV enzyme immunoassay were each compared with a multiplex RT-PCR ELISA by testing 635 nasopharyngeal aspirates collected from children aged < 16 years who had been hospitalised with acute respiratory tract infection during the epidemic season 2002-2003. In this study, the sensitivity of the Directigen Flu A+B assay was unacceptably low (29.3% and 10.0%, respectively) for the detection of influenza A and B viruses. The sensitivity of the Abbott TestPack RSV assay (77.4%) was acceptable and in agreement with the multiplex RT-PCR ELISA.
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Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza B/aislamiento & purificación , Nasofaringe/microbiología , Juego de Reactivos para Diagnóstico , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Reacción en Cadena de la Polimerasa/métodos , Estudios Prospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: A multiplex reverse transcription (RT) polymerase chain reaction combined with a microwell hybridization assay (m-RT-PCR-ELISA) was previously developed to detect nine different microorganisms: enterovirus (EV), influenza virus type A (IVA) and type B (IVB), respiratory syncytial virus (RSV), parainfluenzavirus type 1 (PIV1) and type 3 (PIV3), adenovirus (AV), Mycoplasma pneumoniae (Mpn), Chlamydia pneumoniae (Cpn) in a single test. These organisms do not usually colonize the respiratory tract of humans, but, if present, it may be assumed they are involved in respiratory disease. OBJECTIVES AND STUDY DESIGN: The m-RT-PCR-ELISA was tested on (i) culture supernatants of unknown contents, (ii) by determining the analytical sensitivity of 10-fold serial dilutions of culture supernatants and (iii) by determining clinical sensitivity in a retrospective study on 411 clinical specimens. The specimens were re-tested in parallel by m-RT-PCR-ELISA versus the gold standard culture and immunfluorescence, and versus individual RT-PCR. RESULTS: (i) The 9-valent m-RT-PCR-ELISA shows 83% to 100% concordant results on 103 culture supernatants containing different organisms. (ii) The analytical sensitivity was as follows: higher sensitivity of the 9-valent m-RT-PCR-ELISA in comparison to culture in the cases of PIV3, IVA and IVB (factor 10) and AV and EV (factor 100), and lower sensitivity in case of RSV and PIV1 (factor 10). (iii) The agreement with the gold standard in the kappa statistic was excellent for RSV (kappa = 0.937), IVA (kappa = 0.940), very good for PIV1 (kappa = 0.914), IVB (kappa = 0.907) and satisfactory for PIV3 (kappa = 0.410). For AV, EV and Mpn the m-RT-PCR-ELISA preliminary could be qualified as very good, based on the data derived on culture supernatants. Information about the validity for Cpn is limited. CONCLUSION: The m-RT-PCR-ELISA is a feasible, sensitive and specific method for detection of a broad spectrum of organisms. It is suitable for individual as well as epidemiological diagnosis.
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Infecciones del Sistema Respiratorio/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Adenoviridae/genética , Adenoviridae/aislamiento & purificación , ADN Viral/genética , ADN Viral/aislamiento & purificación , Enterovirus/genética , Enterovirus/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza B/genética , Virus de la Influenza B/aislamiento & purificación , Variaciones Dependientes del Observador , Reproducibilidad de los Resultados , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/aislamiento & purificación , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/microbiología , Respirovirus/genética , Respirovirus/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
The analysis of gene polymorphisms has started to become an interesting field of new services to be provided by clinical laboratories for both, clinical research and routine determinations. To expand the use of these methods and to increase the benefit for patients and the society, cheap and reliable methods for genotyping also allowing for high-turnover analysis need to be established in clinical laboratories. The presented investigation was performed to develop and evaluate a fast real-time PCR method for the detection of three different allele mutations of Cytochrome P450 isoenzyme 2C8 (CYP2C8*2, *3, and *4), which have been demonstrated to influence drug metabolism (and thus the efficacy) of a variety of drugs. The DNA of 122 Caucasian subjects (56 male, 66 female, age (mean +/- STD): 50 +/- 16 years) was analyzed for these mutations by means of classical RFLP-PCR and a new protocol developed for the LightCycler real-time PCR method. The polymorphisms within the CYP2C8 gene were detected by use of two primer pairs and three different pairs of hybridization probes. The results of both methods were perfectly concordant and comparable to results published in the literature (allele frequencies: CYP2C8*2: 0.016, *3: 0.140, *4: 0.074). A subsequent analysis of the related costs revealed comparable resource requirements but substantially longer time needs for RFLP-PCR, resulting in higher overall analysis costs for the older method. In conclusion, the elaborated protocol for real-time PCR analysis of gene mutations CYP2C8*2, *3, and *4 is reliable and cost effective, and thus, suitable for routine laboratory use.
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Hidrocarburo de Aril Hidroxilasas/genética , Farmacogenética/métodos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Hidrocarburo de Aril Hidroxilasas/clasificación , Secuencia de Bases , Citocromo P-450 CYP2C8 , Análisis Mutacional de ADN , Femenino , Fluorescencia , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Isoenzimas/clasificación , Isoenzimas/genética , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Reproducibilidad de los ResultadosRESUMEN
The aim of this study was to generate urgently needed data on respiratory pathogens in German children using an economical and efficient tool. Nasopharyngeal aspirates of hospitalized children 0-16 years of age with an acute respiratory tract infection were tested by a nine-valent multiplex reverse-transcriptase polymerase chain reaction. Of 1281 children, 449 (35%) had an acute respiratory tract infection caused by at least one of the organisms studied; there were 29 cases of dual infection. At least 34-42% of severe acute respiratory tract infections in children under 5 years of age were caused by viruses. In children over 5 years of age, this proportion was 23% (P<0.001). Infection during the first 2 years of life was most frequently due to respiratory syncytial virus (n = 162 cases). Parainfluenza virus type 3 (n = 22) and type 1 (n = 14) were detected almost exclusively in children under 5 years of age. Influenza A (n = 90) and adenoviruses (n = 98) were prevalent in all age groups. The frequency of influenza B virus isolation (n = 17) rose significantly after the age of 5 years. Mycoplasma pneumoniae infection (n = 24 cases, 5.2%) was most frequent in 5- to 16-year-old patients. Only one case of Chlamydia pneumoniae infection was found. Since the distribution of pathogens within the different types of lower respiratory tract infections is very similar, it seems that host factors determine which form of lower respiratory tract infection develops in an individual patient. The multiplex reverse-transcriptase polymerase chain reaction may, in the future, become an important tool for epidemiological studies as well as for individual diagnosis.
Asunto(s)
Chlamydophila pneumoniae/aislamiento & purificación , Infecciones del Sistema Respiratorio/epidemiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virus/aislamiento & purificación , Adolescente , Distribución por Edad , Niño , Preescolar , Infecciones por Chlamydophila/epidemiología , Infecciones por Chlamydophila/microbiología , Chlamydophila pneumoniae/clasificación , Chlamydophila pneumoniae/genética , Alemania , Hospitalización , Humanos , Lactante , Recién Nacido , Mycoplasma pneumoniae/aislamiento & purificación , Nasofaringe/microbiología , Nasofaringe/virología , Neumonía por Mycoplasma/epidemiología , Neumonía por Mycoplasma/microbiología , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , Estaciones del Año , Virosis/virología , Virus/clasificación , Virus/genéticaRESUMEN
Acute respiratory tract infections (ARIs) are leading causes of morbidity and, in developing countries, mortality in children. A multiplex reverse transcription-PCR (RT-PCR) assay was developed to allow in one test the detection of nine different microorganisms (enterovirus, influenza A and B viruses, respiratory syncytial virus [RSV], parainfluenzaviruses type 1 and type 3, adenovirus, Mycoplasma pneumoniae, and Chlamydia pneumoniae) that do not usually colonize the respiratory tracts of humans but, if present, must be assumed to be the cause of respiratory disease. Clinical samples from 1,118 children admitted to the Department of Pediatrics because of an ARI between November 1995 and April 1998 were used for a first clinical evaluation. Detection of one of the microorganisms included in the assay was achieved for 395 of 1,118 (35%) clinical samples, of which 37.5% were RSV, 20% were influenza A virus, 12.9% were adenovirus, 10.6% were enterovirus, 8.1% were M. pneumoniae, 4.3% were parainfluenzavirus type 3, 3.5% were parainfluenzavirus type 1, 2.8% were influenza B virus, and 0.2% were C. pneumoniae. Seasonal variations in the rates of detection of the different organisms were observed, as was expected from the literature. The levels of concordance with the data obtained by commercially available enzyme immunoassays were 95% for RSV and 98% for influenza A. The results show that the multiplex RT-PCR-enzyme-linked immunosorbent assay is a useful and rapid diagnostic tool for the management of children with ARI. Studies of the overall benefit of this method with regard to the use of antibiotics, the use of diagnostic procedures including additional microbiological tests, and hospitalization rate and duration are warranted.
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Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Enfermedad Aguda , Adenoviridae/genética , Adenoviridae/aislamiento & purificación , Niño , Preescolar , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/aislamiento & purificación , ADN Bacteriano/análisis , Enterovirus/genética , Enterovirus/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Estudios de Factibilidad , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/aislamiento & purificación , Virus de la Parainfluenza 1 Humana/genética , Virus de la Parainfluenza 1 Humana/aislamiento & purificación , ARN Viral/análisisRESUMEN
At least two transcription factors, aTFB and aTFA, are required for accurate and faithful in vitro transcription of homologous templates in cell-free extracts from the methanogenic Archaeon Methanococcus thermolithotrophicus. We have recently shown that the function of aTFB can be replaced by eucaryal TATA-binding proteins. Here we demonstrate using template commitment experiments that promoter recognition in an Archaeon is mediated by transcription factors. The archaeal TATA box was identified as recognition site for binding of aTFB by gel shift analyses. aTFB binds also to the TATA box of adenovirus 2 major late promoter suggesting homology of eucaryal and archaeal TATA boxes. Our analyses provide evidence for a common molecular mechanism of transcription initiation by eucaryal RNA polymerases and archaeal RNA polymerase. They indicate also an evolutionary homology for aTFB and TBP.