Daniel Guerendiain

Formalin fixed paraffin embedded (FFPE) material is amenable to HPV detection by the Xpert® HPV assay

BACKGROUND The Xpert(®) HPV Assay (Cepheid(®), Sunnyvale, USA) is a rapid, cartridge-based HPV test validated for use on cervical cytology samples. However, there is an increasing demand for HPV annotation of formalin fixed paraffin embedded (FFPE) material. OBJECTIVES The aim of this study was to determine the suitability of the Xpert HPV assay for the detection of nucleic acid (NA) derived from FFPE samples. STUDY DESIGN A total of 88, 10 μm sections derived from FFPE tissue blocks were assessed, 74 originated from oropharyngeal squamous cell carcinomas (OPSCC) and 14 from a range of other sites. All had previously been tested with a sensitive Luminex(®) based assay with a component also tested with p16 immunohistochemistry (IHC). NA was extracted from samples using the easyMag(®) platform and after dilution was added directly to the Xpert cartridge. Agreement between assays was assessed. RESULTS Overall agreement between the assays was 92%; with a Kappa for HR-HPV detection of 0.833 (95% CI 0.725-0.953). In the 50 samples that had been annotated for p16 status overall agreement between the Xpert assay and the p16 IHC was 90%. CONCLUSIONS These data indicate that FFPE material is amenable to HPV detection by the Xpert assay. To our knowledge, this is the first study to interrogate the use of the Xpert(®) HPV assay for this application.

Underestimation of Staphylococcus aureus (MRSA and MSSA) carriage associated with standard culturing techniques: One third of carriers missed

Objectives Nasal carriers of Staphylococcus (S.) aureus (MRSA and MSSA) have an increased risk for healthcare-associated infections. There are currently limited national screening policies for the detection of S. aureus despite the World Health Organization’s recommendations. This study aimed to evaluate the diagnostic performance of molecular and culture techniques in S. aureus screening, determine the cause of any discrepancy between the diagnostic techniques, and model the potential effect of different diagnostic techniques on S. aureus detection in orthopaedic patients. Methods Paired nasal swabs for polymerase chain reaction (PCR) assay and culture of S. aureus were collected from a study population of 273 orthopaedic outpatients due to undergo joint arthroplasty surgery. Results The prevalence of MSSA nasal colonization was found to be between 22.4% to 35.6%. The current standard direct culturing methods for detecting S. aureus significantly underestimated the prevalence (p = 0.005), failing to identify its presence in approximately one-third of patients undergoing joint arthroplasty surgery. Conclusion Modelling these results to national surveillance data, it was estimated that approximately 5000 to 8000 S. aureus surgical site infections could be prevented, and approximately

Evaluation of Staphylococcus aureus eradication therapy in orthopaedic surgery

140 million to

Detection of Norovirus by BD MAX™, Xpert® Norovirus, and xTAG® Gastrointestinal Pathogen Panel in stool and vomit samples

950 million (approximately £110 million to £760 million) saved in treatment costs annually in the United States and United Kingdom combined, by using alternative diagnostic methods to direct culture in preoperative S. aureus screening and eradication programmes. Cite this article: S. T. J. Tsang, M. P. McHugh, D. Guerendiain, P. J. Gwynne, J. Boyd, A. H. R. W. Simpson, T. S. Walsh, I. F. Laurenson, K. E. Templeton. Underestimation of Staphylococcus aureus (MRSA and MSSA) carriage associated with standard culturing techniques: One third of carriers missed. Bone Joint Res 2018;7:79–84. DOI: 10.1302/2046-3758.71.BJR-2017-0175.R1.

Evaluation of point of care testing platform (ePLEX) for respiratory viral diagnosis

Purpose. Despite WHO recommendations, there is currently no national screening and eradication policy for the detection of methicillin‐sensitive Staphylococcus aureus (MSSA) in the UK prior to elective orthopaedic surgery. This study aimed to evaluate the effectiveness of current standard methicillin‐resistant S. aureus (MRSA) eradication therapies in the context of S. aureus (both MRSA and MSSA) decolonization in an elective orthopaedic population. Methodology. A total of 100 patients awaiting joint replacement surgery who were positive for S. aureus on PCR nasal screening underwent the current standard MRSA pre‐operative decolonization regimen for 5 days. Prior to commencement of the eradication therapy, swabs of the anterior nares, throat and perineum were taken for culture. Further culture swabs were taken at 48–96 h following treatment, at hospital admission for surgery and at hospital discharge. Following the completion of treatment, patients were asked to provide feedback on their experience using Likert rating scales. The primary outcome of this study was S. aureus clearance 48–96 h following eradication treatment. Results/Key Findings. Clearance of S. aureus 48–96 h following treatment was 94 % anterior nares, 66 % throat and 88 % groin. Mean completion with nasal mupirocin was 98 %. There was no statistically significant recolonization effect between the end of the eradication treatment period and the day of surgery (P>0.05) at a median time of 10 days. Conclusion. Current MRSA decolonisation regimens are well tolerated and effective for MSSA decolonization for the anterior nares and groin. The decolonization effect is preserved for at least 10 days following treatment.

Underestimation of Staphylococcus aureus carriage associated with standard culturing techniques

BACKGROUND Norovirus is a leading cause of infectious gastroenteritis, characterized by outbreaks of diarrhoea and vomiting in closed settings. Nucleic acid amplification tests allow rapid and sensitive laboratory diagnosis of norovirus, with a number of commercial platforms now available. OBJECTIVES Evaluate the performance of the Becton Dickinson BD-MAX™System, Cepheid Xpert® Norovirus Assay, and Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) for norovirus detection in stool. Assess the performance of the Xpert® Norovirus Assay and BD-MAX™ in vomit samples. STUDY DESIGN 163 diarrhoeal stool samples were tested on four diagnostic systems (laboratory-defined real time RT-PCR (assigned as gold standard), BD MAX™, Xpert® Norovirus Assay, and xTAG® GPP). A further 70 vomit samples were tested on the Xpert and BD MAX platforms. RESULTS In stool, sensitivity and specificity of the BD-MAX™ was 96.8% and 100%, for Xpert® Norovirus Assay was 91.9% and 100%, and for xTAG® GPP was 79.0% and 87.1%. In vomit samples positive and negative percent agreement was 95.6% and 92.0%, between the BD-MAX™ and Xpert® Norovirus. CONCLUSIONS The BD-MAX™ System with user defined settings and the Xpert® Norovirus Assay showed acceptable sensitivity and specificity for detection of norovirus from stool and vomit. The xTAG GPP assay was less reliable for norovirus detection but can detect a number of other clinically useful enteropathogens. Clinical laboratories must consider skill mix, budget, and sample throughput to determine the best fit for their service.

Evaluation of Enigma® MiniLabTM Influenza A/B RSV assay designed for rapid testing and point of care diagnosis

no: 278 Presentation at ESCV 2016: Poster 201 Evaluation of point of care testing platform (ePLEX) for respiratory viral diagnosis Daniel Guerendiain ∗, Laura MacKenzie, K.E. Templeton NHS Lothian, Royal Infirmary of Edinburgh, UK Background: There is an increasing demand on laboratories to deliver respiratory viral diagnosis by molecular methods. Different strategies are explored which include – point of care testing which is simple, requires minimal hands on time, is fast and can be done in ward areas and not in centralised laboratories. Tests considered need to be shown to have good performance. GenMark Diagnostics Inc. (Carlsbad, USA) has developed a respiratory panel assay (RP) for the ePlex system detecting 26 microbes, including 22 virus and 4 (atypical) bacteria in 90 min. The RP cartridge contains all reagents required to run the RP Panel assay. Lysis and nucleic acid extraction, PCR amplification and hybridization-based electrochemical detection occur inside the cartridge, reducing the hands-on-time to less than 1 min per sample. The objective of this study was to compare and study the performance of the new GenMark ePlex assay against the in house real-time PCR, a lab developed test (LDT). Material and methods: 81 nasopharyngeal swabs samples (NPS) in UTM were previously tested by an in-house Real Time PCR. Samples selected contained the following respiratory pathogens: respiratory syncytial virus, influenza A, influenza B, rhinovirus, enterovirus, bocavirus, coronaviruses, metapneumovirus, parainfluenza viruses, Bordetella pertussis and Mycoplasma pneumoniae 32.1% samples were co-infected, even with 4 different organisms. Samples selected were less than 4 months old with only one freeze/thaw cycle. Ct values ranging from 17.11 to 40.39 mean 24.74. 200 l of each NPS sample was added to the ePlex Sample Buffer device, transferred to the RP cartridge and then inserted on the ePlex device. Agreement between the original LDT results and the results obtained with the ePlex assay was assessed as detected or not detected. Additionally 5 successive 1:10 dilutions were performed for 7 different specimens: RSV, influenza A H1N1, influenza B, rhinovirus, bocavirus, Mycoplasma pneumoniae and Bordetella pertussis. Dilutions were tested on both assays to identify and compare the lower limit of detection to the LDT. Results: Total concordance was observed in 91.73% of cases. Only 10 discrepancies were identified. 7 organisms were detected by the ePlex assay and missed by the LDT and 3 organisms were positive detected by the LDT and negative by the ePlex. Discrepancies were repeated in both assays showing same results. Total concordance was observed in 80% of dilutions. 1 dilution 10−4 RSV sample was detected by the ePlex and resulted negative for the LDT. As well 6 samples (10−3–10−5 dilutions) were positive for the LDT and negative for the ePlex assay. B. pertussis did not detect the 2 lower dilutions as a different target gene was in use in ePlex assay. Conclusion: The preliminary evaluation on a small sample set show a very good agreement across a range of pathogens with the GenMark compares in house real-time PCR. The assay was also found to be very simple and easy to perform and would be suitable for a hospital ward or outpatient environment. http://dx.doi.org/10.1016/j.jcv.2016.08.241 Abstract no: 286 Presentation at ESCV 2016: Poster 202no: 286 Presentation at ESCV 2016: Poster 202 Single genetic clades of EV-D68 strains in 2010, 2013, and 2015 in Osaka City, Japan A. Kaida 1,∗, N. Iritani 1, S.P. Yamamoto 1, D. Kanbayashi 1, Y. Hirai 1, U. Kohdera 2, M. Togawa 3, K. Amo 3, M. Shiomi 4, T. Nishigaki 5, T. Kageyama 6, H. Kubo 1 1 Osaka City Institute of Public Health and Environmental Sciences, Japan 2 Nakano Children’s Hospital, Japan 3 Osaka City General Hospital, Japan 4 Aizenbashi Hospital, Japan 5 Osaka Police Hospital, Japan 6 Influenza Virus Research Center, National Institute of Infectious Diseases, Japan Background: Detection of Enterovirus D68 (EV-D68), a cause of acute respiratory tract infection (ARTI), was rarely reported before the early 2000s. Molecular analyses have demonstrated that recently detected EV-D68 strains are of three major genetic clades. We previously reported the emergence of EV-D68 in children with ARTI in Osaka City, Japan in 2010. Objectives: This study surveyed EV-D68 among children with ARTI since its first endemic period in 2010 and conducted molecular analyses of the detected viral genome sequences. Methods: During November 2010–December 2015, 2215 respiratory clinical specimens were obtained from children (<10 years old) with ARTI. Specimens from patients diagnosed with influenza were excluded. Real-time RT-PCR was used to detect enteroviruses. Viral protein 4 (VP4) or VP1 genes were sequenced to identify EV-