Resolving Discrepancies in Idylla BRAF Mutational Assay Results Using Targeted Next-Generation Sequencing.

BRAF mutation identification is important for the diagnosis and treatment of several tumor types, both solid and hematologic. Rapid identification of BRAF mutations is required to determine eligibility for targeted BRAF inhibitor therapy. The Idylla BRAF mutation assay is a rapid, multiplex allele-specific PCR test designed to detect the most common oncogenic BRAF V600 mutations in formalin-fixed paraffin-embedded (FFPE) tissue samples. Here, we describe the validation of the Idylla BRAF mutation assay in our laboratory. During routine clinical practice, we noticed cases in which BRAF V600 mutations were identified with unusual amplification curves, with three cases displaying a delayed amplification within a double amplification pattern and two false-positive calls. We therefore initiated a quality improvement effort to systematically and retrospectively evaluate next-generation sequencing (NGS)-tested cases with BRAF mutations identified within five amino acids of BRAF codon V600 and did not identify additional false-positive cases. We hypothesize that late amplification in a double amplification pattern may represent non-specific amplification, whereas cases displaying single delayed amplification curves may stem from the presence of either non-V600 variants, very low-level V600 variants, cytosine deamination artifacts, and/or non-specific amplification by an allele-specific PCR primer. Regardless, we recommend that Idylla BRAF cases with non-classical amplification curves undergo reflex NGS testing. These findings are likely relevant for other Idylla assays interrogating hotspot mutations in genes such as EGFR, IDH1/2, KRAS, and NRAS.

Rapid molecular identification of Rana dybowskii by species-specific primers.

Oviductus Ranae is the dried oviduct from Rana dybowskii, a forest frog species with medicinal, tonic, and cosmetic properties. Due to the high price and resource shortage, counterfeit varieties of Oviductus Ranae often appear in the market. However, traditional identification methods cannot accurately differentiate between Oviductus Ranae and its adulterants. In this study, a rapid molecular identification method has been established. The method involves extracting genomic DNA in just 30 s using filter paper purification, species-specific rapid polymerase chain reaction (PCR) amplification, and finally, fluorescence detection of the products. It can accurately identify Oviductus Ranae and its three common adulterants in about 30 min, making the process simple, fast, and highly specific.

Analysis of Microbiomes from Ultra-Low Biomass Surfaces Using Novel Surface Sampling and Nanopore Sequencing.

The rapid assessment of microbiomes from ultra-low biomass environments such as cleanrooms or hospital operating rooms has a number of applications for human health and spacecraft manufacturing. Current techniques often employ lengthy protocols using short-read DNA sequencing technology to analyze amplified DNA and have the disadvantage of a longer analysis time and lack of portability. Here, we demonstrate a rapid (~24 hours) on-site nanopore-based sequencing approach to characterize the microbiome of a NASA Class 100K cleanroom where spacecraft components are assembled. This approach employs a modified protocol of Oxford Nanopore's Rapid PCR Barcoding Kit in combination with the recently developed Squeegee-Aspirator for Large Sampling Area (SALSA) surface sampling device. Results for these ultra-low biomass samples revealed DNA amplification ~1 to 2 orders of magnitude above process control samples and were dominated primarily by Paracoccus and Acinetobacter species. Negative control samples were collected to provide critical data on background contamination, including Cutibacerium acnes, which most likely originated from the sampling reagents-associated microbiome (kitome). Overall, these results provide data on a novel approach for rapid low-biomass DNA profiling using the SALSA sampler combined with modified nanopore sequencing. These data highlight the critical need for employing multiple negative controls, along with using DNA-free reagents and techniques, to enable a proper assessment of ultra-low biomass samples.

A Disposable Electromagnetic Bi-Directional Micropump Utilizing a Rotating Multi-Pole Ring Magnetic Coupling.

Electromagnetic bi-directional micropumps (EMBM) are indispensable for the development of portable devices which enable fluid transportation in forward and reverse directions. However, the high disposal cost of rare-earth magnets attached to the fluidic part and the low pump density due to multiple motors limit their practical application in disposable multi-channel microfluidic applications such as droplet-based oscillatory-flow (DBOF) rapid PCR amplification. Therefore, this paper presented a low-cost, disposable, high-pump-density EMBM. To reduce the disposal cost, we separated the magnets from the disposable fluidic part and used cylindrical holes to store and guide the magnet, which resulted in the ability to reuse all the magnets. To increase the pump density, we used the combination of one motor and one multi-pole ring magnet to drive several channels simultaneously. A proof-of-concept prototype with a pump density of 0.28 cm-2 was fabricated and experimentally evaluated. The fabricated micropump exhibited a maximum flow rate of 0.86 mL/min and a maximum backpressure of 0.5 kPa at a resonant frequency around 50 Hz. The developed multi-channel micropump with reusable magnets is highly beneficial to the development of low-cost and high-throughput rapid PCR amplification microchips and therefore can have a significant impact on timely infectious disease recognition and intervention.

A Portable Droplet Magnetofluidic Device for Point-of-Care Detection of Multidrug-Resistant Candida auris.

Candida auris is an emerging multidrug-resistant fungal pathogen that can cause severe and deadly infections. To date, C. auris has spurred outbreaks in healthcare settings in thirty-three countries across five continents. To control and potentially prevent its spread, there is an urgent need for point-of-care (POC) diagnostics that can rapidly screen patients, close patient contacts, and surveil environmental sources. Droplet magnetofluidics (DM), which leverages nucleic acid-binding magnetic beads for realizing POC-amenable nucleic acid detection platforms, offers a promising solution. Herein, we report the first DM device-coined POC.auris-for POC detection of C. auris. As part of POC.auris, we have incorporated a handheld cell lysis module that lyses C. auris cells with 2 min hands-on time. Subsequently, within the palm-sized and automated DM device, C. auris and control DNA are magnetically extracted and purified by a motorized magnetic arm and finally amplified via a duplex real-time quantitative PCR assay by a miniaturized rapid PCR module and a miniaturized fluorescence detector-all in ≤30 min. For demonstration, we use POC.auris to detect C. auris isolates from 3 major clades, with no cross reactivity against other Candida species and a limit of detection of ∼300 colony forming units per mL. Taken together, POC.auris presents a potentially useful tool for combating C. auris.

Rapid polymerase chain reaction test for the early diagnosis of gonococcal keratoconjunctivitis.

PURPOSES: A case of gonococcal keratoconjunctivitis rapidly diagnosed by a vaginal swab PCR Xpert® CT/NG assay. METHODS: Case report. RESULTS: A 26-year-old woman presented to our emergency department with severe perilimbal stromal melting in both eyes and profuse purulent discharge for one day. Upon emergent ocular consultation, gonococcal keratoconjunctivitis was suspected. A vaginal swab was sent for rapid PCR Xpert® CT/NG assay which reported positive Neisseria gonorrhoeae (NG) and Chlamydia Trachoma (CT) DNA detection within 90 min. Due to the rapid diagnosis, adequate medical intervention with ceftriaxone injection was administered. Gonococcal keratitis with stromal melting was stabilized within 5 days of presentation. The patient was discharged with complete epithelial healing by the 8th day. However, 10 weeks after discharge, inadvertent rubbing of the left eye resulted in corneal perforation with iris prolapse. Lamellar keratoplasty with corneal patch graft was performed with amniotic membrane grafting. Xpert® CT/NG assay was performed again with conjunctival swab for recurring mild eye discharge. Both NG and CT were negative. The patient thus stabilized with no further complications. CONCLUSIONS: Rapid stromal melting can occur with un-diagnosed or delayed diagnosis of gonorrhea with ocular involvement. Speedy and accurate diagnosis by the highly sensitive and specific Xpert® CT/NG assay can provide early definite diagnosis for prompt treatment in prevention of gonococcal infection induced corneal perforations.

Determination of honey adulterated with corn syrup by quantitative amplification of maize residual DNA using ultra-rapid real-time PCR.

BACKGROUND: Honey is a naturally sweet syrup made by honeybees from floral nectar. However, high-fructose corn syrup has been prevalently used for the adulteration of honey. A novel molecular method was developed for the characterization of corn syrup-adulterated honey by specific amplification and quantification of maize residual DNA in honey. An ultra-rapid real-time polymerase chain reaction (UR-qPCR) system for rapid amplification and protocol for direct purification of residual DNA from honey were described. RESULTS: Rapidity of maize DNA amplification was acquired within 20 min for a limit of detection of around three copies of targeted DNAs. The amplification of maize residual DNA in honeys adulterated with corn syrup from 5% to 80% (v/v) showed that a minimum rate of 10% adulteration can be identified, and Maize genomic DNA in 5 mL of adulterated honeys was from 13 ± 9 copies to 2478 ± 827 copies, respectively. However, the residual DNA of maize was also detected in natural honey produced in the region where pollen and nectar of maize were collected, and the quantity of maize genomic DNA in these natural honeys was in the range of 10% adulteration with corn syrup. Therefore, detection of both pollen and residual DNA of maize in honey is important in identifying the source of maize residual DNA present in honey. CONCLUSION: A rapid PCR assay was first developed for the accurate detection and quantification of maize residual DNA in honey. It is a useful tool for specific identification of the corn syrup used for honey adulteration. Further studies on residual DNA in various types of corn syrup and specificity of primer are recommended. © 2021 Society of Chemical Industry.

Rapid diagnosis of SARS-CoV-2 pneumonia on lower respiratory tract specimens.

BACKGROUND: The ongoing SARS-CoV-2 pandemic requires the availability of accurate and rapid diagnostic tests, especially in such clinical settings as emergency and intensive care units. The objective of this study was to evaluate the diagnostic performance of the Vivalytic SARS-CoV-2 rapid PCR kit in lower respiratory tract (LRT) specimens. METHODS: Consecutive LRT specimens (bronchoalveolar lavage and bronchoaspirates) were collected from Intensive Care Units of San Martino Hospital (Genoa, Italy) between November 2020 and January 2021. All samples underwent RT-PCR testing by means of the Allplex™ SARS-CoV-2 assay (Seegene Inc., South Korea). On the basis of RT-PCR results, specimens were categorized as negative, positive with high viral load [cycle threshold (Ct) ≤ 30] and positive with low viral load (Ct of 31-35). A 1:1:1 ratio was used to achieve a sample size of 75. All specimens were subsequently tested by means of the Vivalytic SARS-CoV-2 rapid PCR assay (Bosch Healthcare Solutions GmbH, Germany). The diagnostic performance of this assay was assessed against RT-PCR through the calculation of accuracy, Cohen's κ, sensitivity, specificity and expected positive (PPV) and negative (NPV) predictive values. RESULTS: The overall diagnostic accuracy of the Vivalytic SARS-CoV-2 was 97.3% (95% CI: 90.9-99.3%), with an excellent Cohen's κ of 0.94 (95% CI: 0.72-1). Sensitivity and specificity were 96% (95% CI: 86.5-98.9%) and 100% (95% CI: 86.7-100%), respectively. In samples with high viral loads, sensitivity was 100% (Table 1). The distributions of E gene Ct values were similar (Wilcoxon's test: p = 0.070), with medians of 35 (IQR: 25-36) and 35 (IQR: 25-35) on Vivalytic and RT-PCR, respectively (Fig. 1). NPV and PPV was 92.6% and 100%, respectively. Table 1 Demographic characteristics and data sample type of the study cases (N = 75) Male, N (%) 56 (74.6%) Age (yr), Median (IQR) 65 (31-81) BAS, N (%) 43 (57.3%)  Negative 30.2%  Positive-High viral load [Ct ≤ 30] 27.9%  Positive-Low viral load [Ct 31-35] 41.9% BAL, N (%) 32 (42.7%)  Negative 37.5%  Positive-High viral load [Ct ≤ 30] 40.6%  Positive-Low viral load [Ct 31-35] 21.9% Data were expressed as proportions for categorical variables. Specimens were categorized into negative, positive with high viral load [cycle threshold (Ct) ≤ 30] and positive with low viral load (Ct of 31-35). BAS bronchoaspirates, BAL bronchoalveolar lavage, Ct cycle threshold Fig. 1 Distribution of E gene cycle threshold values of the rapid PCR and RT-PCR CONCLUSIONS: Vivalytic SARS-CoV-2 can be used effectively on LRT specimens following sample liquefaction. It is a feasible and highly accurate molecular procedure, especially in samples with high viral loads. This assay yields results in about 40 min, and may therefore accelerate clinical decision-making in urgent/emergency situations.

Rapid PCR powered by microfluidics: A quick review under the background of COVID-19 pandemic.

PCR has been widely used in different fields including molecular biology, pathogen detection, medical diagnosis, food detection and etc. However, the difficulty of promoting PCR in on-site point-of-care testing reflects on challenges relative to its speed, convenience, complexity, and even cost. With the emerging state-of-art of microfluidics, rapid PCR can be achieved with more flexible ways in micro-reactors. PCR plays a critical role in the detection of SARS-CoV-2. Under this special background of COVID-19 pandemic, this review focuses on the latest rapid microfluidic PCR. Rapid PCR is concluded in two main features, including the reactor (type, size, material) and the implementation of thermal cycling. Especially, the compromise between speed and sensitivity with microfluidic PCR is explored based on the system ratio of (thermal cycling time)/(reactor size). Representative applications about the detection of pathogens and SARS-CoV-2 viruses based on rapid PCR or other isothermal amplification are discussed as well.

Ultrafast and Real-Time Nanoplasmonic On-Chip Polymerase Chain Reaction for Rapid and Quantitative Molecular Diagnostics.

Advent and fast spread of pandemic diseases draw worldwide attention to rapid, prompt, and accurate molecular diagnostics with technical development of ultrafast polymerase chain reaction (PCR). Microfluidic on-chip PCR platforms provide highly efficient and small-volume bioassay for point-of-care diagnostic applications. Here we report ultrafast, real-time, and on-chip nanoplasmonic PCR for rapid and quantitative molecular diagnostics at point-of-care level. The plasmofluidic PCR chip comprises glass nanopillar arrays with Au nanoislands and gas-permeable microfluidic channels, which contain reaction microchamber arrays, a precharged vacuum cell, and a vapor barrier. The on-chip configuration allows both spontaneous sample loading and microbubble-free PCR reaction during which the plasmonic nanopillar arrays result in ultrafast photothermal cycling. After rapid sample loading less than 3 min, two-step PCR results for 40 cycles show rapid amplification in 264 s for lambda-DNA, and 306 s for plasmids expressing SARS-CoV-2 envelope protein. In addition, the in situ cyclic real-time quantification of amplicons clearly demonstrates the amplification efficiencies of more than 91%. This PCR platform can provide rapid point-of-care molecular diagnostics in helping slow the fast-spreading pandemic.

The development of miniSTRs as a method for high-speed direct PCR.

There are situations in which it would be very valuable to have a DNA profile within a short time; for example, in mass disasters or airport security. In previous work, we have promoted reduced size STR amplicons for the analysis of degraded DNA. We also noticed that shorter amplicons are more robust during amplification, making them inhibition resistant, and potentially applicable to high-speed direct PCR. Here, we describe a set of miniSTRs capable of rapid direct PCR amplification. The selected markers are a subset of the Combined DNA Index System (CODIS) loci modified to permit high-speed amplification. Using the proposed protocol, the amplification of eight loci plus amelogenin directly from a saliva sample can be completed in 7 min and 38 s using a two-step PCR with 30 cycles of 98°C for 2 s and 62°C for 7 s on a Streck Philisa thermocycler. Selection of DNA polymerase, optimization of the two-step PCR cycling conditions, the primer concentrations, and the dilution of saliva is described. This method shows great potential as a quick screening method to obtain a presumptive DNA profile when time is limited, particularly when combined with high-speed separation and detection methods.

Development and evaluation of a novel quenching probe PCR (GENECUBE) assay for rapidly detecting and distinguishing between Chlamydia pneumoniae and Chlamydia psittaci.

Early detection of the family Chlamydiaceae as pathogens is essential worldwide for the rapid and sufficient management of atypical pneumonia. GENECUBE (TOYOBO) is a novel fully automated gene analyzer capable of amplifying and detecting target DNAs within 50 min. In this study, we developed a new PCR assay with a specific quenching probe (PCR-QC assay) for rapidly distinguishing between Chlamydia pneumoniae (CPN) and Chlamydia psittaci (CPS). The PCR-QC assay enabled us to precisely and simultaneously detect the 2 different types of DNA fragments even in a mixed sample by identifying unique melting temperatures. Next, we examined a total of 300 frozen samples from patients with respiratory tract infection using the PCR-QC assay and the cell culture method as the gold standard. Kappa index for agreement between the PCR-QC assay and the culture method was 0.43 (95% confidential interval (CI): 0.08-0.78). The sensitivity and specificity of the PCR-QC assay were 36.3% (4/11; 95% CI: 10.9-69.2%)) and 99.0% (286/289; 95% CI: 97.0-99.8%), respectively. The samples positive for CPN (n = 13) or CPS (n = 1) by either method were also examined by a conventional PCR TaqMan assay, which produced the same results as those from the PCR-QC assay. Furthermore, the PCR-QC assay using GENECUBE shortened the full detection time for CPN or CPS (within 50 min vs. more than 2 to 3 h) compared with conventional PCR TaqMan assays. Therefore, the new PCR-QC assay system equipped with GENECUBE is useful for rapidly detecting CPN or CPS pathogens in clinical laboratory, and may improve the management of atypical pneumonia.

Clinical impact of rapid influenza PCR in the adult emergency department on patient management, ED length of stay, and nosocomial infection rate.

BACKGROUND: Seasonal influenza causes significant morbidity and mortality and incurs large economic costs. Influenza like illness is a common presenting concern to Emergency Departments (ED), and optimizing the diagnosis of influenza in the ED has the potential to positively affect patient management and outcomes. Therapeutic guidelines have been established to identify which patients most likely will benefit from anti-viral therapy. OBJECTIVES: We assessed the impact of rapid influenza PCR testing of ED patients on laboratory result generation and patient management across two influenza seasons. METHODS: A pre-post study was performed following a multifaceted clinical redesign including the implementation of rapid influenza PCR at three diverse EDs comparing the 2016-2017 and 2017-2018 influenza seasons. Testing parameters including turn-around-time and diagnostic efficiency were measured along with rates of bed transfers, hospital-acquired (HA) influenza, and ED length of stay (LOS). RESULTS: More testing of discharged patients was performed in the post-intervention period, but influenza rates were the same. Identification of influenza-positive patients was significantly faster, and there was faster and more appropriate prescription of anti-influenza medication. There were no differences in bed transfer rates or HA influenza, but ED LOS was reduced by 74 minutes following clinical redesign. CONCLUSIONS: Multifaceted clinical redesign to optimize ED workflow incorporating rapid influenza PCR testing can be successfully deployed across different ED environments. Adoption of rapid influenza PCR can streamline testing and improve antiviral stewardship and ED workflow including reducing LOS. Further study is needed to determine if other outcomes including bed transfers and rates of HA influenza can be affected by improved testing practices.

Relevance of reviewing endpoint analysis for negative results on the Xpert Xpress Flu/RSV.

After the implementation of the Xpert Xpress Flu/respiratory syncytial virus (RSV) assay for rapid respiratory molecular testing, we investigated the significance of reported endpoint values for influenza A, influenza B, and RSV). This study prospectively analyzed nasopharyngeal swabs submitted to our virology laboratory in the 2018/19 influenza season. Initial testing was performed on the Xpress Flu/RSV assay. Samples were further tested on a laboratory-developed multiplex polymerase chain reaction (laboratory-developed multiplex respiratory test [LDT]) if the sample was reported as negative by the Xpress Flu/RSV but had an elevated endpoint value ≥5 for any respiratory virus target. There were 1040 negative results on the Xpress Flu/RSV; thirty-one had at least one endpoint value ≥5 [influenza A (25), influenza B (1), RSV (2), influenza A/RSV (1), and influenza A/B/RSV (2)]. Five samples (5/31, 16.1%) were positive on the LDT for influenza A or RSV. In contrast, the positivity rate on the LDT for negative Xpress Flu/RSV samples with endpoint values less than 5 was 0.35% (P < .0001). A threshold for endpoint values could not reliably be established to differentiate a potential influenza A positive result from a negative result on the LDT. Routine evaluation ofendpoint values should be a consideration for laboratories implementing Xpress Flu/RSV, in addition to supplementary respiratory virus testing for clinically relevant situations.

Combining Procalcitonin and Rapid Multiplex Respiratory Virus Testing for Antibiotic Stewardship in Older Adult Patients With Severe Acute Respiratory Infection.

OBJECTIVES: Virus infection is underevaluated in older adults with severe acute respiratory infections (SARIs). We aimed to evaluate the clinical impact of combining point-of-care molecular viral test and serum procalcitonin (PCT) level for antibiotic stewardship in the emergency department (ED). DESIGN: A prospective twin-center cohort study was conducted between January 2017 and March 2018. SETTING AND PARTICIPANTS: Older adult patients who presented to the ED with SARIs received a rapid molecular test for 17 respiratory viruses and a PCT test. MEASURES: To evaluate the clinical impact, we compared the outcomes of SARI patients between the experimental cohort and a propensity score-matched historical cohort. The primary outcome was the proportion of antibiotics discontinuation or de-escalation in the ED. The secondary outcomes included duration of intravenous antibiotics, length of hospital stay, and mortality. RESULTS: A total of 676 patients were included, of which 169 patients were in the experimental group and 507 patients were in the control group. More than one-fourth (27.9%) of the patients in the experimental group tested positive for virus. Compared with controls, the experimental group had a significantly higher proportion of antibiotics discontinuation or de-escalation in the ED (26.0% vs 16.1%, P = .007), neuraminidase inhibitor uses (8.9% vs 0.6%, P < .001), and shorter duration of intravenous antibiotics (10.0 vs 14.5 days, P < .001). CONCLUSIONS AND IMPLICATIONS: Combining rapid viral surveillance and PCT test is a useful strategy for early detection of potential viral epidemics and antibiotic stewardship. Clustered viral respiratory infections in a nursing home is common. Patients transferred from nursing homes to ED may benefit from this approach.

Evaluation of three consecutive versions of a commercial rapid PCR test to screen for methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Screening for methicillin-resistant Staphylococcus aureus (MRSA) is part of many recommendations to control MRSA. Several rapid PCR tests are available commercially and updated versions are constantly released. We aimed to evaluate the performance of three consecutive versions (G3, Gen3 and NxG) of the XpertMRSA test. METHODS: Routine samples for MRSA screening were simultaneously tested by culture and rapid PCR. The three versions of XpertMRSA were used successively and compared with culture. RESULTS: A total of 3512, 2794 and 3288 samples were analysed by culture and by the G3, Gen3 and NxG XpertMRSA versions, respectively. The rates of positive-by-culture in the three groups were 5.0%, 4.7% and 4.3%, respectively. The sensitivity improved over time (71.4, 95% CI 64.0-77.9; 82.3, 95% CI 74.4-88.2; and 84.3%, 95% CI 77.0-89.7, respectively), but not significantly. The specificity (98.4, 95% CI 97.9-98.8; 96.8, 95% CI 96.0-97.4; and 99.1, 95% CI 98.7-99.4, respectively) and the positive likelihood ratios (45.7, 95% CI 34.4-60.8; 25.6, 95% CI 20.5-32.0; and 97.1, 95% CI 66.3-142.4) were significantly lower in the Gen3 version (p < 0.00001). CONCLUSIONS: These significant differences in performance show the importance of evaluating each new version of a commercial test.

Targeted management of influenza A/B outbreaks incorporating the cobas® Influenza A/B & RSV into the virology laboratory.

During the 2017/18 influenza season, the authors' virology laboratory implemented the cobas® Influenza A/B & RSV (Roche Molecular Diagnostics, Pleasanton, CA, USA) for influenza outbreak management in two scenarios: initial outbreak investigation or at outbreak conclusion to avoid prolonged measures. Twenty-seven investigations were conducted, including declaration of 11 influenza A/B outbreaks. Thirty percent of investigations would have missed the standard batched daily laboratory-developed respiratory polymerase chain reaction (PCR), and delayed outbreak confirmation until the following day. The average reduction in turnaround time for influenza A/B testing was 10.2 h. A rapid molecular PCR in specific outbreak scenarios improved timely management of influenza outbreaks.

Impact of Rapid Molecular Diagnostic Testing of Respiratory Viruses on Outcomes of Adults Hospitalized with Respiratory Illness: a Multicenter Quasi-experimental Study.

A standard multiplex PCR offers comprehensive testing for respiratory viruses. However, it has traditionally been performed in a referral laboratory with a lengthy turnaround time, which can reduce patient flow through the hospital. We aimed to determine whether the introduction of a rapid PCR, but with limited targets (Cepheid Xpert Flu/RSV XC), was associated with improved outcomes for adults hospitalized with respiratory illness. A controlled quasi-experimental study was conducted across three hospitals in New South Wales, Australia. Intervention groups received standard multiplex PCR during the preimplementation, July to December 2016 (n = 953), and rapid PCR during the postimplementation, July to December 2017 (n = 1,209). Control groups (preimplementation, n = 937, and postimplementation, n = 1,102) were randomly selected from adults hospitalized with respiratory illness during the same periods. The outcomes were hospital length of stay (LOS) and microbiology test utilization (blood culture, urine culture, sputum culture, and respiratory bacterial and virus serologies). The introduction of rapid PCR was associated with a nonsignificant 8.9-h reduction in median LOS (95% confidence interval [CI], -21.5 h to 3.7 h; P = 0.17) for all patients and a significant 21.5-h reduction in median LOS (95% CI, -36.8 h to -6.2 h; P < 0.01) among patients with positive test results in an adjusted difference-in-differences analysis. For patients receiving test results before disposition, rapid PCR use was associated with a significant reduction in LOS, irrespective of test results. Compared with standard PCR testing, rapid PCR use was significantly associated with fewer blood culture (adjusted odds ratio [aOR], 0.67; 95% CI, 0.5 to 0.82; P < 0.001), sputum culture (aOR, 0.56; 95% CI, 0.47 to 0.68, P < 0.001), bacterial serology (aOR, 0.44; 95% CI, 0.35 to 0.55, P < 0.001) and viral serology (aOR, 0.42; 95% CI, 0.33 to 0.53, P < 0.001) tests, but not with fewer urine culture tests (aOR, 0.94; 95% CI, 0.78 to 1.12, P = 0.48). Rapid PCR testing of adults hospitalized with respiratory illnesses can deliver benefits to patients and reduce resource utilization. Future research should consider a formal economic analysis and assess its potential impacts on clinical decision making.

A fast and visual method for duplex shrimp pathogens detection with high specificity using rapid PCR and molecular beacon.

Molecular diagnosis of genome is one of the major methods for pathogens detection. The commonly used PCR method can realize an exponential amplification of the target gene but is time-consuming. In this work, we proposed a duplex and visual method using rapid PCR combined with molecular beacons to specifically detect two kinds of shrimp pathogens in one reaction tube. We only need to observe the fluorescence change of the reaction tube with naked eye to determine the result. A home-made automatic transfer equipment allows reaction tubes shuttling quickly between two water baths to achieve rapid PCR amplification. A simple device was also designed to present the detection results easily determined with naked eye. This duplex and visual detection method is fast, low-cost and of high specificity. From DNA extraction to results judgment, only 15 min was enough. Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and Vibrio parahaemolyticus (VP) are two common shrimp pathogens which were chosen as our detection objects. This method may give a possibility to conduct end-point visual duplex detection, which may make a positive influence on the pathogen prevention.

Multicenter Evaluation of the ePlex Respiratory Pathogen Panel for the Detection of Viral and Bacterial Respiratory Tract Pathogens in Nasopharyngeal Swabs.

The performance of the new ePlex Respiratory Pathogen (RP) panel (GenMark Diagnostics) for the simultaneous detection of 19 viruses (influenza A virus; influenza A H1 virus; influenza A 2009 H1 virus; influenza A H3 virus; influenza B virus; adenovirus; coronaviruses [HKU1, OC43, NL63, and 229E]; human rhinovirus/enterovirus; human metapneumovirus; parainfluenza viruses 1, 2, 3, and 4; and respiratory syncytial virus [RSV] [RSV subtype A and RSV subtype B]) and 2 bacteria (Mycoplasma pneumoniae and Chlamydia pneumoniae) was evaluated. Prospectively and retrospectively collected nasopharyngeal swab (NPS) specimens (n = 2,908) were evaluated by using the ePlex RP panel, with the bioMérieux/BioFire FilmArray Respiratory Panel (BioFire RP) as the comparator method. Discordance analysis was performed by using target-specific PCRs and bidirectional sequencing. The reproducibility of the assay was evaluated by using reproducibility panels comprised of 6 pathogens. The overall agreement between the ePlex RP and BioFire RP results was >95% for all targets. Positive percent agreement with the BioFire RP result for viruses ranged from 85.1% (95% confidence interval [CI], 80.2% to 88.9%) to 95.1% (95% CI, 89.0% to 97.9%), while negative percent agreement values ranged from 99.5% (95% CI, 99.1% to 99.7%) to 99.8% (95% CI, 99.5% to 99.9%). Additional testing of discordant targets (12%; 349/2,908) confirmed the results of ePlex RP for 38% (131/349) of samples tested. Reproducibility was 100% for all targets tested, with the exception of adenovirus, for which reproducibilities were 91.6% at low virus concentrations and 100% at moderate virus concentrations. The ePlex RP panel offers a new, rapid, and sensitive "sample-to-answer" multiplex panel for the detection of the most common viral and bacterial respiratory pathogens.