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A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously-diagnosed A phagocytophilum cases. The sensitivity and rapidness of this assay represent a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.Q fever, caused by Coxiella burnetii, is a worldwide zoonotic disease that may cause severe forms in humans and requires a specific and prolonged antibiotic treatment. Although current serological and molecular detection tools allow a reliable diagnosis of the disease, culture of C. burnetii strains is mandatory to assess their susceptibility to antibiotics and sequence their genome in order to optimize patient management and epidemiological studies. However, cultivating this fastidious microorganism is difficult and restricted to reference centers as it requires biosafety-level 3 laboratories and relies on cell culture performed by experienced technicians. In addition, the culture yield is low, which results in a small number of isolates being available. In this work, we developed a novel high content screening (HCS) isolation strategy based on optimized high-throughput cell culture and automated microscopic detection of infected cells with specifically-designed algorithms targeting cytopathic effects. This method was more efficient than the shell-vial assay, at the level of time dependency, when applied to both frozen specimens (7 isolates recovered by HCS only, sensitivity 91% vs 78% for shell-vial) and fresh samples (1 additional isolate using HCS, sensitivity 7% vs 5% for shell-vial), for which most strains were recovered more rapidly with the new technique. In addition, detecting positive cultures by an automated microscope reduced the need for expertise and saved 24% of technician working time. Application of HCS to antibiotic susceptibility testing of 12 strains demonstrated that it was as efficient as the standard procedure that combines shell-vial culture and quantitative PCR. Copyright © 2020 American Society for Microbiology.Accurate detection of influenza A virus (IAV) is crucial for patient management, infection control, and epidemiological surveillance. The World Health Organization and the Centers for Disease Control and Prevention have recommended using the M gene as the diagnostic gene target for reverse-transcription-PCR (RT-PCR). However, M gene RT-PCR has reduced sensitivity for recent IAV due to novel gene mutations. Here, we sought to identify novel diagnostic targets for IAV RT-PCR assay using long-read third-generation sequencing. Direct nanopore sequencing from 18 nasopharyngeal specimens and one saliva specimen showed that the 5' and 3' ends of the PB2 gene and the entire NS gene were highly abundant. Primers selected for PB2 and NS genes were well-matched with seasonal or avian IAV gene sequences. Our novel PB2 and NS gene real-time RT-PCR assays showed similar or lower limit of detection than M gene RT-PCR, and achieved 100% sensitivity and specificity in the detection of A(H1N1) and A(H3N2), and A(H7N9) in nasopharyngeal and saliva specimens. For 10 patients with IAV M gene RT-PCR conversion in sequentially-collected specimens, NS and/or PB2 gene RT-PCR was positive in 2 (20%) initial specimens that were missed by M gene RT-PCR. M4205 In conclusion, we have shown that PB2 or NS gene RT-PCRs are suitable alternatives to the recommended M gene RT-PCR for diagnosis of IAV. Long-read nanopore sequencing facilitates identification of novel diagnostic targets. Copyright © 2020 American Society for Microbiology.The QIAstat-Dx Respiratory Panel (QIAstat-Dx RP) is a multiplex in vitro diagnostic test for the qualitative detection of 20 pathogens directly from nasopharyngeal swab (NPS) specimens. The assay is a performed using a simple sample to answer platform with results available in approximately 69 minutes. The pathogens identified are adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, human metapneumovirus A+B, influenza A, influenza A H1, influenza A H3, influenza A H1N1/2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, rhinovirus/enterovirus, respiratory syncytial virus A+B, Bordetella pertussis, Chlamydophila pneumoniae and Mycoplasma pneumoniae This multicenter evaluation provides data obtained from 1994 prospectively collected and 310 retrospectively collected (archived) NPS specimens with performance compared to the BioFire FilmArray Respiratory Panel version 1.7. The overall percent agreement between QIAstat-Dx RP and the comparator testing was 99.5%. In the prospective cohort, the QIAstat-Dx RP demonstrated a positive percent agreement of 94.0% or greater for detection of all but four analytes coronaviruses 229E, NL63 and OC43, and rhinovirus/enterovirus. The test also demonstrated a negative percent agreement of ≥ 97.9% for all analytes. The QIAstat-Dx RP is a robust and accurate assay for rapid, comprehensive testing for respiratory pathogens. Copyright © 2020 Leber et al.Infections due to methicillin-resistant Staphylococcus aureus (MRSA) are present worldwide and represent a major public health concern. Capability of PCR followed by high-resolution melt (HRM) curve analysis for detection of community-associated and livestock-associated MRSA strains and identification of Staphylococcal protein A (spa) locus was evaluated in 74 MRSA samples which were isolated from the environment, humans and pigs on a single piggery. PCR-HRM curve analysis identified four spa types among MRSA samples and differentiated MRSA strains accordingly. A non-subjective differentiation model was developed according to genetic confidence percentage values produced by tested samples, which did not require visual interpretation of HRM curve results. The test was carried out at different settings and result data was re-analysed and confirmed with DNA sequencing. PCR-HRM curve analysis proved to be a robust and reliable test for spa typing and can be used as a tool in epidemiological studies. Copyright © 2020 American Society for Microbiology.M4205