No abstract

Four human coronaviruses (HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OC43) are associated with a range of respiratory outcomes, including bronchiolitis and pneumonia. Their epidemiologies and clinical characteristics are poorly described and are often reliant on case reports. To address these problems, we conducted a large-scale comprehensive screening for all four coronaviruses by analysis of 11,661 diagnostic respiratory samples collected in Edinburgh, United Kingdom, over 3 years between July 2006 and June 2009 using a novel four-way multiplex real-time reverse transcription-PCR (RT-PCR) assay. Coronaviruses were detected in 0.3 to 0.85% of samples in all age groups. Generally, coronaviruses displayed marked winter seasonality between the months of December and April and were not detected in summer months, which is comparable to the pattern seen with influenza viruses. HCoV-229E was the exception; detection was confined to the winter of 2008 and was sporadic in the following year. There were additional longer-term differences in detection frequencies between seasons, with HCoV-OC43 predominant in the first and third seasons and HCoV-HKU1 dominating in the second (see Results for definitions of seasons). A total of 11 to 41% of coronaviruses detected were in samples testing positive for other respiratory viruses, although clinical presentations of coronavirus monoinfections were comparable to those of viruses which have an established role in respiratory disease, such as respiratory syncytial virus, influenza virus, and parainfluenza viruses. The novel multiplex assay for real-time pan-coronavirus detection enhances respiratory virus diagnosis, overcomes potential diagnostic problems arising through seasonal variation in coronavirus frequency, and provides novel insights into the epidemiology and clinical implications of coronaviruses.Four human coronaviruses (human coronavirus 229E [HCoV-229E], HCoV-HKU1, HCoV-NL63, and HCoV-OC43) are associated with a range of respiratory symptoms, including highmorbidity outcomes such as pneumonia and bronchiolitis (26,31,35). Specifically, HCoV-NL63 has been associated with croup (33) and HCoV-HKU1 with febrile convulsion (18). Coronaviruses are frequently codetected with other respiratory viruses, particularly with human respiratory syncytial virus (HRSV) (17). Whether coronaviruses contribute to disease severity in such coinfections is currently unclear. Other coronaviruses infecting humans include human enteric coronavirus, which is closely related to HCoV-OC43 and is associated with necrotizing enterocolitis and gastroenteritis (10,27).Coronaviruses are globally distributed (7,32,34,38), although there are differences in the frequency of detection of the four viruses in different parts of the world at different times (6,11,15,16,22,28,29). Longitudinal studies of coronavirus epidemiology are lacking in the literature and are restricted to descriptions representing a maximum of 1 year for all four respiratory coronaviruses or 2 years for three coronaviruses (9,17,18). Th...

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitablefor high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory.

Laboratory diagnosis of viral respiratory infections is generally performed by virus isolation in

The combined real-time PCR assay is more sensitive for diagnosis of the main viruses and atypical bacteria that cause CAP compared with conventional methods, and obtains results in a clinically relevant time period.