The three unprecedented outbreaks of emerging human coronavirus (HCoV) infections at the beginning of the twentyfirst century have highlighted the necessity for readily available, accurate and fast diagnostic testing methods. The laboratory diagnostic methods for human coronavirus infections have evolved substantially, with the development of novel assays as well as the availability of updated tests for emerging ones. Newer laboratory methods are fast, highly sensitive and specific, and are gradually replacing the conventional gold standards. This presentation reviews the current laboratory methods available for testing coronaviruses by focusing on the coronavirus disease 2019 (COVID-19) outbreak going on in Wuhan. Viral pneumonias typically do not result in the production of purulent sputum. Thus, a nasopharyngeal swab is usually the collection method used to obtain a specimen for testing. Nasopharyngeal specimens may miss some infections; a deeper specimen may need to be obtained by bronchoscopy. Alternatively, repeated testing can be used because over time, the likelihood of the SARS-CoV-2 being present in the nasopharynx increases. Several integrated, random-access, point-of-care molecular devices are currently under development for fast and accurate diagnosis of SARS-CoV-2 infections. These assays are simple, fast and safe and can be used in the local hospitals and clinics bearing the burden of identifying and treating patients.

A set of broad-range PCR primers for the 16S rRNA gene in bacteria were tested, along with three series of oligonucleotide probes to detect the PCR product. The first series of probes is broad in range and consists of a universal bacterial probe, a gram-positive probe, a Bacteroides-Flavobacterium probe, and two probes for other gram-negative species. The second series was designed to detect PCR products from seven major bacterial species or groups frequently causing meningitis: Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, Escherichia coli and other enteric bacteria, Listeria monocytogenes, and Staphylococcus aureus. The third series was designed for the detection of DNA from species or genera commonly considered potential contaminants of clinical samples, including cerebrospinal fluid (CSF): BaciUus, Corynebacterium, Propionibacterium, and coagulase-negative Staphylococcus spp. The primers amplified DNA from all 124 different species of bacteria tested. Southern hybridization testing of the broad-range probes with washes containing 3 M tetramethylammonium chloride indicated that this set of probes correctly identified all but two of the 102 bacterial species tested, the exceptions being Deinococcus radiopugnans and Gardnerella vaginalis. The gram-negative and gram-positive probes hybridized to isolates of two newly characterized bacteria, AUloiococcus otitis and Rochalimaea henselii, as predicted by Gram stain characteristics. The CSF pathogen and contaminant probe sequences were compared with available sequence information and with sequencing data for 32 different species. Testing of the CSF pathogen and contaminant probes against DNA from over 60 different strains indicated that, with the exception of the coagulase-negative Staphylococcus probes, these probes provided the correct identification of bacterial species known to be found in CSF.

48 Background. Nucleic acid amplification tests (NAATs) are the primary means of 49 identifying acute infections caused by severe acute respiratory syndrome coronavirus 2 50 (SARS-CoV-2). Accurate and fast test results may permit more efficient use of protective and 51 isolation resources and allow for rapid therapeutic interventions. 52 Methods. We evaluated the analytical and clinical performance characteristics of the Xpert ® 53 Xpress SARS-CoV-2 (Xpert) test, a rapid, automated molecular test for SARS-CoV-2. 54 Analytical sensitivity and specificity/interference were assessed with infectious SARS-CoV-2, 55 other infectious coronavirus species including SARS-CoV, and 85 nasopharyngeal swab 56 specimens positive for other respiratory viruses including endemic human coronaviruses 57 (hCoVs). Clinical performance was assessed using 483 remnant upper and lower respiratory 58 specimens previously analyzed by standard of care (SOC) NAATs. 59 Results. The limit of detection of the Xpert test was 0.01 plaque forming units (PFU)/mL. 60 Other hCoVs, including Middle East Respiratory Syndrome coronavirus, were not detected by 61 the Xpert test. SARS-CoV, a closely related species in the Sarbecovirus subgenus, was 62 detected by a broad-range target (E) but was distinguished from SARS-CoV-2 (SARS-CoV-2-63 specific N2 target). Compared to SOC NAATs, the positive agreement of the Xpert test was 64 219/220 (99.5%) and the negative agreement was 250/261 (95.8%). A third tie-breaker 65 NAAT resolved all but three of the discordant results in favor the Xpert test. 66 Conclusions. The Xpert test provided sensitive and accurate detection of SARS-CoV-2 in a 67 variety of upper and lower respiratory tract specimens. The high sensitivity and fast time to 68 results of approximately 45 minutes may impact patient management. 69 70 Laboratory diagnosis of infections caused by severe acute respiratory syndrome coronavirus 2 72 (SARS-CoV-2) is usually accomplished by performing nucleic acid amplification tests 73 (NAATs) on respiratory tract specimens. An antibody response is often not detected in the 74 first week to ten days of symptoms and antibody testing is therefore generally unhelpful for 75 acute diagnosis(1-3), with virus isolation in culture presenting significant biosafety risks. 76 Upper respiratory tract (URT) specimens such as nasopharyngeal swabs (NPS) and 77 oropharyngeal swabs (OPS) generally have high SARS-CoV-2 viral loads upon symptom 78 onset.(2, 4-6) URT specimens may also have detectable RNA during the pre-symptomatic 79 period(7), and pediatric patients who remain asymptomatic through the entire course of 80 on June 9, 2020 by guest http://jcm.asm.org/ Downloaded from 4 infection can persistently shed RNA in URT specimens for two weeks or longer.(4, 8) 81 Importantly, NPS may have higher viral loads than OPS.(6) Lower respiratory tract (LRT) 82 specimens including sputum(7, 9) and tracheal aspirates(10) (TA) are often positive for RNA 83 early in disease and remain positive longer than URT sources.(5) 84 NAATs are...

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Sepsis is a major cause of morbidity, mortality, and increased medical expense. Rapid diagnosis improves outcomes and reduces costs. The FilmArray blood culture identification panel (BioFire Diagnostics LLC, Salt Lake City, UT), a highly multiplexed PCR assay, can identify 24 etiologic agents of sepsis (8 Gram-positive, 11 Gram-negative, and 5 yeast species) and three antimicrobial resistance genes (mecA, vanA/B, and blaKPC) from positive blood culture bottles. It provides results in about 1 h with 2 min for assay setup. We present the results of an eight-center trial comparing the sensitivity and specificity of the panel with those of the laboratories' standard phenotypic identification techniques, as well as with molecular methods used to distinguish Acinetobacter baumannii from other members of the A. calcoaceticus-A. baumannii complex and to detect antimicrobial resistance genes. Testing included 2,207 positive aerobic blood culture samples, 1,568 clinical and 639 seeded. Samples were tested fresh or were frozen for later testing within 8 h after the bottles were flagged as positive by an automated blood culture system. At least one organism was detected by the panel in 1,382 (88.1%) of the positive clinical specimens. The others contained primarily off-panel organisms. The panel reported multiple organisms in 81 (5.86%) positive clinical specimens. The unresolved blood culture identification sensitivity for all target detections exceeded 96%, except for Klebsiella oxytoca (92.2%), which achieved 98.3% sensitivity after resolution of an unavoidable phenotypic error. The sensitivity and specificity for vanA/B and blaKPC were 100%; those for mecA were 98.4 and 98.3%, respectively.