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• Developing and validation of a loop-mediated isothermal amplification for clinical diagnosis of WNV. • Amplifications on extracted viral RNA and on crude samples with Thermal Cycler and Pebble Device. • RT-LAMP for WNV detection can be used at point of care in hospital setting or small laboratories. • RT-LAMP assay is an efficient consistent technique for detecting WNV. • RT-lamp requires extracted RNA and have lower sensitivity when run directly on crude material. West Nile Virus (WNV) causes a serious public health concern in many countries around the world. Virus detection in pathological samples is a key component of WNV infection diagnostic, classically performed by real-time PCR. In outbreak situation, rapid detection of the virus, in peripheral laboratories or at point of care, is crucial to guide decision makers and for the establishment of adequate action plans to prevent virus dissemination. Here, we evaluate a Loop-mediated isothermal amplification (LAMP) tool for WNV detection. Amplifications were performed comparatively on extracted viral RNA and on crude samples using a classical thermal cycler and a portable device (pebble device). qRT-PCR was used as gold standard and two sets of urine samples (n = 62 and n = 74) were used to evaluate the retained amplification protocols and assess their sensitivity and specificity. RT-LAMP on RNA extracts and crude samples showed a sensitivity of 90 % and 87 %, respectively. The specificity was 100 % for extracts and 97 % for crude samples. Using the device, the RT-LAMP on extracted RNA was comparable to the gold standard results (100 % sensitivity and specificity) and it was a bit lower on crude samples (65 % sensitivity and 94 % specificity). These results show that RT-LAMP is an efficient technique to detect WNV. RT-LAMP provides a rapid, sensitive, high-throughput and portable tool for accurate WNV detection and has potentials to facilitate diagnostic and surveillance efforts both in the laboratory and in the field, especially in developing countries. [ABSTRACT FROM AUTHOR]

Abstract: Background: The quality of clinical specimens is a crucial determinant for virological diagnosis. Objectives: We compared the viral diagnostic yield for influenza A and respiratory syncytial virus (RSV) from the recently developed nasopharyngeal flocked swabs (NPFS) with nasopharyngeal aspirates (NPA) collected in parallel from 196 hospitalized children with acute respiratory infection during the peak period of influenza A and RSV activity in Hong Kong. Specimens were tested by RT-PCR for influenza A and RSV and viral load determined. They were also tested by direct immunofluorescence (DIF) for influenza A and B, RSV, parainfluenza types 1–3 and adenovirus. Results: Both NPA and NPFS had excellent sensitivity (100%) for detecting influenza A by RT-PCR but NPA was slightly more sensitive than NPFS for detecting RSV by both RT-PCR (100% vs. 92.3%) and DIF (87.2% vs. 84.6%) and for detecting influenza A by DIF (90.2% vs. 82.9%). Viral load for influenza A in NPA and NPFS was not significantly different but that for RSV was higher in NPA. Conclusion: NPA remains the optimal specimen for diagnosis of respiratory infections by RT-PCR and DIF. However, collection of NPFS is easier to perform in an out-patient setting, was more acceptable to parents and less likely to generate aerosols than NPA engendering potentially less infection control hazard. [Copyright &y& Elsevier]

Abstract: Background:: The protocols of WHO network laboratories facilitated development of rapid diagnosis for SARS coronavirus (CoV) using reverse transcription (RT)-PCR assays. However, several reports have shown that conventional and real-time PCR assays were very specific for SARS CoV but lack sensitivity depending on the assay, specimen, and time course of disease. Objective:: To evaluate an automatic nucleic acid extraction system and two standardized real-time PCR assays for rapid diagnosis of SARS CoV during outbreak and post-epidemic periods in Hong Kong. Study design:: Specimens from clinically suspected SARS patients collected during outbreak and post-epidemic periods were tested by an automatic nucleic acid extraction system followed by our first generation conventional RT-PCR and two standardized real-time PCR assays (Artus GmbH, Germany and Roche Diagnostics, Germany). Paired serum samples were assayed for increasing titer against SARS CoV. Results:: In the SARS epidemic, Artus and Roche PCR assays exhibited sensitivities of 87% and 85% for respiratory specimens (n=64), 91% and 88% for stool (n=44), and 82% for urine (n=29). A specificity of 100% was exhibited by both PCR assays except Artus attained only a 92% specificity for stool. For post-epidemic period, no SARS CoV was identified among 56 respiratory specimens by all PCR assays. Inhibitors to PCR assays were detected at an average rate of 7–8% among 202 clinical specimens. Conclusion:: This study highlights the high throughput and performance of automatic RNA extraction in coordination with standardized real-time PCR assays suitable for large-scale routine diagnosis in case of future SARS epidemic. As no SARS CoV was detected among specimens collected during post-epidemic period, the positive predictive value of real-time PCR assays for detection of SARS CoV during low epidemic requires further evaluation. [Copyright &y& Elsevier]

• The COVID-19 Ag Respi-Strip assay is a new immunochromatographic diagnostic tool for antigenic diagnosis of SARS-CoV-2. • This test did not reduce significantly the number of samples outsourced for COVID-19 confirmation. • The sensitivity of this rapid test is poor and improvements are needed to enhance its performances. The COVID-19 Ag (Antigen) Respi-Strip assay is a new immunochromatographic diagnostic tool recently available for antigenic diagnosis of SARS-CoV-2. The proposed sensitivity is not higher than 60 %, but its high specificity allows both quick decisions for the management of patients and confirmation by molecular diagnosis for only negative tests. However, from the first tests performed, we suspected that the sensitivity observed with routine use was much lower than that announced by the manufacturer. Over a period of one month, we compared the negative results obtained with the COVID-19 Ag Respi-Strip kit with those obtained from qRT-PCR performed in a laboratory qualified for the molecular diagnosis of SARS-CoV-2. All samples tested were naso-pharyngeal smears from UTM-RT medium. Of 774 patients tested, 714 negative samples were sent for confirmation, and 159 were found to be positive by qRT-PCR. The median positive percentage agreement was 23.9 % (95 % CI: 14.2 %–38.2 %). The Cohen's kappa score was 0.35. Using this immunochromatographic assay as a triage test did not significantly reduce the number of samples outsourced for COVID-19 confirmation by qRT-PCR. In addition, even if the turn-around time is short, the assay is completely manual, which is not suitable for large volumes of routine samples. The sensitivity of this rapid test is poor, and improvements are needed to enhance its performance. [ABSTRACT FROM AUTHOR]

• Detection of SARS-CoV-2 within 3.5 h (sample-to-answer-time) by random access real time PCR. • Adaption of an already published real time PCR for SARS-CoV-2 to a fully automatic, random access PCR system. • Detection of all SARS-CoV (from 2002, which may be soon labelled as SARS-CoV-1, and SARS-CoV-2) with a PCR for the highly conserved E gene region. • Differentiation of SARS-CoV-2 with a PCR for the RdRp Gene. [ABSTRACT FROM AUTHOR]