Application of a portable instrument for rapid and reliable detection of SARS-CoV-2 infection in any environment.

The ongoing outbreak of the novel coronavirus (SARS-CoV-2) infection is creating serious challenges for health laboratories that seek to identify viral infections as early as possible, optimally at the earliest appearance of symptom. Indeed, there is urgent need to develop and deploy robust diagnostic methodologies not only to use in health laboratory environments but also directly in places where humans circulate and spread the virus such as airports, trains, boats, and any public aggregation places. The success of a reliable and sensitive asymptomatic diagnosis relies on the identification and measurement of informative biomarkers from human host and virus in a rapid, sensitive, and inexpensive manner. The objective of this article is to describe an innovative multidisciplinary approach to develop an efficient, inexpensive, and easy-to-use portable instrument (bCUBE® by Hyris Ltd) that can be employed as a surveillance system for the emergency caused by SARS-CoV-2. A solution for Coronavirus testing, compliant with CDC guidelines, is scheduled to be released in the next weeks. In addition, we will describe a workflow and path of an integrated multi-omic approach that will lead to host and pathogen biomarker discovery in order to train the instrument to provide reliable results based on a specific biomarker's fingerprint of SARS-CoV-2 infection.

CXCL13 in laboratory diagnosis of Lyme neuroborreliosis-the performance of the recomBead and ReaScan CXCL13 assays in human cerebrospinal fluid samples.

The chemokine CXCL13 is used as complement to serology in the diagnostics of Lyme neuroborreliosis (LNB). We evaluated and compared the semi-quantitative, cassette-based ReaScan CXCL13 assay with the quantitative recomBead CXCL13 assay using a collection of 209 cerebrospinal fluid samples. The categorical agreement between results interpreted as negative, grey zone, and positive by the two methods was 87%. The diagnostic sensitivity was higher using the recomBead assay, whereas specificity was higher using ReaScan. Few manual steps, and a short turn-around time with no batching of samples makes the ReaScan CXCL13 assay an attractive complement to serology in the diagnostics of LNB.

Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

Epidemiology of bloodstream Candida species in a Spanish tertiary care hospital as a guide for implementation of T2MR (T2CANDIDA®) for rapid diagnosis of candidemia.

The aim of this work was to study the epidemiology of candidemia in our hospital in order to determine whether the T2MR system might be a useful tool for early diagnosis of candidemia in selected units. We perform a retrospective review of all candidemia episodes registered in the last 12 years in selected units of our hospital in adult and pediatric patients. Candida species and antifungal susceptibility patterns were registered. A total of 686 isolates were registered, of which 625 were infections due to the five most common species of Candida. C. albicans (45.6%) and C. parapsilosis (33.1%) were the predominant species found in our institution. In adults these species were closely followed by C. glabrata (12-21%) in all units. While in pediatric medical and intensive care units (PICU), these species were followed by other uncommon yeasts. Resistance rates to triazoles were low in C. albicans and C. parapsilosis. In C. glabrata and C. tropicalis the resistance rates to fluconazole ranged from 10.86 to 6.67%. Resistance rates for echinocandins were very low and all strains were susceptible to amphotericin B. T2Candida® might be useful to guide antifungal targeted treatment and discontinuation of antifungal empirical treatment in those units where the five most common Candida species represent more than the ninety percent of the isolates. The selection of medical and surgical units should be based on local epidemiology and antifungal susceptibility patterns. Incidence should be taken into account in order to make clinical decisions based on negative results. LAY ABSTRACT: T2Candida® might be useful selectively in clinical units according to their local epidemiology, antifungal resistance patterns, and incidence of candidemia. It optimizes the clinical value of positive results supporting decisions about targeted therapies or discontinuations based on negative results.

The Alzheimer's Association international guidelines for handling of cerebrospinal fluid for routine clinical measurements of amyloid ß and tau.

The core cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers amyloid beta (Aß42 and Aß40), total tau, and phosphorylated tau, have been extensively clinically validated, with very high diagnostic performance for AD, including the early phases of the disease. However, between-center differences in pre-analytical procedures may contribute to variability in measurements across laboratories. To resolve this issue, a workgroup was led by the Alzheimer's Association with experts from both academia and industry. The aim of the group was to develop a simplified and standardized pre-analytical protocol for CSF collection and handling before analysis for routine clinical use, and ultimately to ensure high diagnostic performance and minimize patient misclassification rates. Widespread application of the protocol would help minimize variability in measurements, which would facilitate the implementation of unified cut-off levels across laboratories, and foster the use of CSF biomarkers in AD diagnostics for the benefit of the patients.

SARS-CoV-2 lgM/lgG antibody detection confirms the infection after three negative nucleic acid detection.

An ongoing outbreak of viral pneumonia was caused by a novel coronavirus in China in 2019. By March 19, over 200 thousand confirmed cases of SARS-CoV-2 infection and over 9000 deaths have been reported throughout the world. For this infectious disease, nucleic acid detection is still the gold standard for pathogenic detection. However, nucleic acid detection takes a long time and has relatively high "false negative"; therefore, we need urgently a convenient and accurate detection method to make up for this deficiency. In this article, we will show such technical characteristics of lgM/lgG serum antibody detection, compared with nucleic acid detection.

Open Development and Clinical Validation of Multiple 3D-Printed Nasopharyngeal Collection Swabs: Rapid Resolution of a Critical COVID-19 Testing Bottleneck.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a severe international shortage of the nasopharyngeal swabs that are required for collection of optimal specimens, creating a critical bottleneck blocking clinical laboratories' ability to perform high-sensitivity virological testing for SARS-CoV-2. To address this crisis, we designed and executed an innovative, cooperative, rapid-response translational-research program that brought together health care workers, manufacturers, and scientists to emergently develop and clinically validate new swabs for immediate mass production by 3D printing. We performed a multistep preclinical evaluation of 160 swab designs and 48 materials from 24 companies, laboratories, and individuals, and we shared results and other feedback via a public data repository (http://github.com/rarnaout/Covidswab/). We validated four prototypes through an institutional review board (IRB)-approved clinical trial that involved 276 outpatient volunteers who presented to our hospital's drive-through testing center with symptoms suspicious for COVID-19. Each participant was swabbed with a reference swab (the control) and a prototype, and SARS-CoV-2 reverse transcriptase PCR (RT-PCR) results were compared. All prototypes displayed excellent concordance with the control (κ = 0.85 to 0.89). Cycle threshold (CT ) values were not significantly different between each prototype and the control, supporting the new swabs' noninferiority (Mann-Whitney U [MWU] test, P > 0.05). Study staff preferred one of the prototypes over the others and preferred the control swab overall. The total time elapsed between identification of the problem and validation of the first prototype was 22 days. Contact information for ordering can be found at http://printedswabs.org Our experience holds lessons for the rapid development, validation, and deployment of new technology for this pandemic and beyond.

Design of engineered surfaces for prospective detection of SARS-CoV-2 using quartz crystal microbalance-based techniques.

INTRODUCTION: Rapid transmission of the severe acute respiratory syndrome coronavirus 2 has affected the whole world and forced it to a halt (lockdown). A fast and label-free detection method for the novel coronavirus needs to be developed along with the existing enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR)-based methods. AREAS COVERED: In this report, biophysical aspects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein are outlined based on its recent reported electron microscopy structure. Protein binding sites are analyzed theoretically, which consisted of hydrophobic and positive charged amino acid residues. Different strategies to form mixed self-assembled monolayers (SAMs) of hydrophobic (CH3) and negatively charged (COOH) groups are discussed to be used for the specific and strong interactions with spike protein. Bio-interfacial interactions between the spike protein and device (sensor) surface and its implications toward designing suitable engineered surfaces are summarized. EXPERT OPINION: Implementation of the engineered surfaces in quartz crystal microbalance (QCM)-based detection techniques for the diagnosis of the novel coronavirus from oral swab samples is highlighted. The proposed strategy can be explored for the label-free and real-time detection with sensitivity up to ng level. These engineered surfaces can be reused after desorption.

Evaluation of the Risk of Laboratory Microbial Contamination during Routine Testing in Automated Clinical Chemistry and Microbiology Laboratories.

BACKGROUND: Every clinical specimen is potentially infectious, but data regarding risk for contamination of the laboratory environment during routine testing are scarce. We assessed contamination during routine sample analysis in automated clinical chemistry and microbiology laboratories. METHODS: A fluorescent marker was applied to specimen container exteriors to assess the impact of gross contamination. Nonpathogenic MS2 virus was added to remnant blood, urine, and ESwab matrices as a biomarker of cross-contamination. Samples were processed and analyzed using Roche Cobas 8100 and ISE, c502, e602, and c702 modules (blood) and BD Kiestra total laboratory automation (blood, urine, ESwabs) over 3 experiments. Fluorescence transfer to laboratory surfaces and personnel was visualized using ultraviolet light. Surfaces were swabbed and assessed for MS2 cross-contamination by RT-PCR. Adherence to standard precautions by laboratory staff was assessed by observation. RESULTS: Fluorescence was observed on 49 of 165 (30%) laboratory surfaces and personnel and 21 of 93 (23%) total laboratory automation instruments. Fluorescence transferred most frequently to gloves (31/40), computer accessories (9/18), and specimen loading racks (12/12). None of 123 areas swabbed were positive for MS2. Improper personal protective equipment use occurred at a rate of 0.36 and 0.15 events per staff per hour in the chemistry and microbiology laboratories, respectively. Hand-washing compliance was observed for 61 of 132 (46%) staff members evaluated. CONCLUSIONS: Analysis of grossly contaminated specimens on automated chemistry and microbiology equipment elicits a low likelihood of instrument contamination. However, handling contaminated specimen containers can result in contamination of environmental laboratory surfaces, representing a source of risk that is heightened by low adherence to appropriate personal protective equipment.

Evaluation of Contamination Risk by the cobas e 602 Serology Module Before Viral Load Testing on the cobas 6800 System.

BACKGROUND: Diagnosis of HCV, HBV, and HIV involves antibody screening followed by confirmation and/or treatment decision using nucleic acid tests. However, minimal data exist evaluating the risk of nucleic acid cross-contamination on serology devices upstream of molecular testing despite the potential clinical and laboratory workflow advantages of single specimen vial testing for both procedures. METHODS: We conducted a checkerboard study investigating the potential risk of HCV, HBV, and HIV nucleic acid cross-contamination on 480 negative specimens by a serology screening instrument that uses disposable tips for sample transfer, rather than a fixed needle, before molecular testing. RESULTS: Nucleic acid contamination was observed in 0 of 480 negative specimens when processed with alternating high-titer HCV, HBV, or HIV specimens on the serology platform. CONCLUSIONS: This study suggests that specimens analyzed by a serology instrument using disposable tips for sample transfer may be suitable for direct primary specimen reflex testing by a sensitive nucleic acid confirmatory test.

FISH and chips: a review of microfluidic platforms for FISH analysis.

Fluorescence in situ hybridization (FISH) allows visualization of specific nucleic acid sequences within an intact cell or a tissue section. It is based on molecular recognition between a fluorescently labeled probe that penetrates the cell membrane of a fixed but intact sample and hybridizes to a nucleic acid sequence of interest within the cell, rendering a measurable signal. FISH has been applied to, for example, gene mapping, diagnosis of chromosomal aberrations and identification of pathogens in complex samples as well as detailed studies of cellular structure and function. However, FISH protocols are complex, they comprise of many fixation, incubation and washing steps involving a range of solvents and temperatures and are, thus, generally time consuming and labor intensive. The complexity of the process, the relatively high-priced fluorescent probes and the fairly high-end microscopy needed for readout render the whole process costly and have limited wider uptake of this powerful technique. In recent years, there have been attempts to transfer FISH assay protocols onto microfluidic lab-on-a-chip platforms, which reduces the required amount of sample and reagents, shortens incubation times and, thus, time to complete the protocol, and finally has the potential for automating the process. Here, we review the wide variety of approaches for lab-on-chip-based FISH that have been demonstrated at proof-of-concept stage, ranging from FISH analysis of immobilized cell layers, and cells trapped in arrays, to FISH on tissue slices. Some researchers have aimed to develop simple devices that interface with existing equipment and workflows, whilst others have aimed to integrate the entire FISH protocol into a fully autonomous FISH on-chip system. Whilst the technical possibilities for FISH on-chip are clearly demonstrated, only a small number of approaches have so far been converted into off-the-shelf products for wider use beyond the research laboratory.

Pandemic printing: a novel 3D-printed swab for detecting SARS-CoV-2.

OBJECTIVES: To design and evaluate 3D-printed nasal swabs for collection of samples for SARS-CoV-2 testing. DESIGN: An iterative design process was employed. Laboratory evaluation included in vitro assessment of mock nasopharyngeal samples spiked with two different concentrations of gamma-irradiated SARS-CoV-2. A prospective clinical study compared SARS-CoV-2 and human cellular material recovery by 3D-printed swabs and standard nasopharyngeal swabs. SETTING, PARTICIPANTS: Royal Melbourne Hospital, May 2020. Participants in the clinical evaluation were 50 hospital staff members attending a COVID-19 screening clinic and two inpatients with laboratory-confirmed COVID-19. INTERVENTION: In the clinical evaluation, a flocked nasopharyngeal swab sample was collected with the Copan ESwab and a mid-nasal sample from the other nostril was collected with the 3D-printed swab. RESULTS: In the laboratory evaluation, qualitative agreement with regard to SARS-CoV-2 detection in mock samples collected with 3D-printed swabs and two standard swabs was complete. In the clinical evaluation, qualitative agreement with regard to RNase P detection (a surrogate measure of adequate collection of human cellular material) in samples collected from 50 hospital staff members with standard and 3D-printed swabs was complete. Qualitative agreement with regard to SARS-CoV-2 detection in three pairs of 3D-printed mid-nasal and standard swab samples from two inpatients with laboratory-confirmed SARS-CoV-2 was also complete. CONCLUSIONS: Using 3D-printed swabs to collect nasal samples for SARS-CoV-2 testing is feasible, acceptable to patients and health carers, and convenient.

Clinical Accuracy of a New Rapid Assay for Fecal Calprotectin Measurement.

BACKGROUND: Fecal calprotectin is an excellent biomarker for distinguishing inflammatory bowel disease from irritable bowel syndrome and for evaluation of disease activity in Crohn's disease and ulcerative colitis. The aim of this work was to evaluate the analytical performance of a new flow immune chromatography assay by comparing it to our standardized laboratory gold standard system. METHODS: A total of 100 stool samples sent for routine calprotectin level measurements were analyzed by the Liaison XL system and the QuantOn Cal assay simultaneously using the same cutoff values for both assays. Performance of the QuantOn Cal assay was assessed by calculating sensitivity, specificity, and accuracy. RESULTS: Compared with the gold standard, the sensitivity, specificity, and accuracy of the QuantOn Cal assay were 98.7%, 76.2%, and 94.0%, respectively. Furthermore, linear correlation of calprotectin levels between the two assays were found to be significant by Pearson's correlation coefficient test (r = 0.82, p-values < 0.0001). CONCLUSIONS: The QuantOn Cal assay demonstrated good performance, both qualitative and quantitative when compared to the Liaison XL system. This novel and rapid assay is well suited for measuring fecal calprotectin as a point of care or home-based assay when laboratory services are limited or not available.

Comparison of bacterial endotoxin testing methods in purified pharmaceutical water matrices.

Current bacterial endotoxin testing systems can be labor-intensive and time-consuming, involving several manual pipetting steps. In our quality control laboratory, annually, we test about 15,000 samples of different grades of purified water, WFI and water samples taken to validate cleaning procedures for endotoxins. We are currently using the Kinetic-QCL™ assay which is a pharmacopeia method that provides reliable results. We compared this assay with another Limulus amebocyte lysate (LAL)-based assay (Endosafe®-MCS) and an alternative endpoint fluorescent recombinant Factor C (rFC) assay (ENDOZYME II GO®). Both these assays have been developed to reduce analyst preparation time. Our objective was to assess if they could increase the throughput of our testing while maintaining low rates of invalid results. The results demonstrated that the two most appropriate methods for rapid endotoxin detection in water are our current assay, K-QCL, and the rFC-based assay, ENDOZYME II GO. This latter assay was found to be less sensitive to interference than our current assay, particularly in cleaning validation water samples. It also showed better performance, accuracy, repeatability and had a shorter time-to-results. ENDOZYME II GO assay allows quick testing of large numbers of samples with reliable results and is a good alternative for conventional LAL assays.

Fractured aluminum nasopharyngeal swab during drive-through testing for COVID-19: radiographic detection of a retained foreign body.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has increased the need for safe and efficient testing as a key containment strategy. Drive-through testing with nasopharyngeal swab has been implemented in many places in the USA as it allows for expeditious testing of large numbers of patients, limits healthcare workers' risk of exposure, and minimizes the use of personal protective equipment. We present a case where the aluminum shaft of the nasopharyngeal swab fractured during specimen collection at a drive-through testing facility and was suspected to have remained in the asymptomatic patient. Initial evaluation with a series of radiographs covering the skull base, neck, chest, and abdomen did not reveal the swab. On further clinical evaluation, the swab was found endoscopically, lodged between the left inferior turbinate and nasal floor, and was removed by an otorhinolaryngologist. Using a phantom model, we aimed to delineate an imaging technique to better visualize the aluminum shaft of the nasopharyngeal swab on radiographs to help in identification. A technique using lower tube voltage (kVp) with tight collimation centered at the nasal bones area produced the best visualization of the aluminum shaft of the swab. Recognition that aluminum foreign bodies may be difficult to visualize radiographically and  optimization of radiograph acquisition technique may help guide clinical management in unusual cases. Further evaluation with computed tomography or endoscopy should be considered in suspected cases where radiographs are negative.

Multicentre comparison of quantitative PCR-based assays to detect SARS-CoV-2, Germany, March 2020.

Containment strategies and clinical management of coronavirus disease (COVID-19) patients during the current pandemic depend on reliable diagnostic PCR assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we compare 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. While most assays performed well, we identified detection problems in a commonly used assay that may have resulted in false-negative test results during the first weeks of the pandemic.

Nasopharyngeal swab collection in the suspicion of Covid-19.

RT-PCR detection of SARS-CoV-2 mRNA on nasopharyngeal swab is the standard for diagnosing active Covid-19 disease in asymptomatic subjects and in symptomatic patients without the typical radiological findings. Nasopharyngeal swabbing appears a trivial procedure, still an inappropriate nasopharyngeal sampling, performed by untrained operators, can be a relevant cause of false negative findings with a clear negative impact on the effort to control the epidemic and, when PPE is not properly used, this can expose healthcare workers and patients to risks of contagion.

A Wearable Stethoscope for Long-Term Ambulatory Respiratory Health Monitoring.

Lung sounds acquired by stethoscopes are extensively used in diagnosing and differentiating respiratory diseases. Although an extensive know-how has been built to interpret these sounds and identify diseases associated with certain patterns, its effective use is limited to individual experience of practitioners. This user-dependency manifests itself as a factor impeding the digital transformation of this valuable diagnostic tool, which can improve patient outcomes by continuous long-term respiratory monitoring under real-life conditions. Particularly patients suffering from respiratory diseases with progressive nature, such as chronic obstructive pulmonary diseases, are expected to benefit from long-term monitoring. Recently, the COVID-19 pandemic has also shown the lack of respiratory monitoring systems which are ready to deploy in operational conditions while requiring minimal patient education. To address particularly the latter subject, in this article, we present a sound acquisition module which can be integrated into a dedicated garment; thus, minimizing the role of the patient for positioning the stethoscope and applying the appropriate pressure. We have implemented a diaphragm-less acousto-electric transducer by stacking a silicone rubber and a piezoelectric film to capture thoracic sounds with minimum attenuation. Furthermore, we benchmarked our device with an electronic stethoscope widely used in clinical practice to quantify its performance.

Determination of Albumin, Glucose, and Creatinine Employing a Single Sequential Injection Lab-at-Valve with Mono-Segmented Flow System Enabling In-Line Dilution, In-Line Single-Standard Calibration, and In-Line Standard Addition.

A mono-segmented sequential injection lab-at-valve (SI-LAV) system for the determination of albumin, glucose, and creatinine, three key biomarkers in diabetes screening and diagnosis, was developed as a single system for multi-analyte analysis. The mono-segmentation technique was employed for in-line dilution, in-line single-standard calibration, and in-line standard addition. This made adjustments to the sample preparation step easy unlike the batch-wise method. The results showed that the system could be used for both fast reaction (albumin) and slow reaction (glucose with enzymatic reaction and creatinine). In the case of slow reaction, the analysis time could be shortened by using the reaction rate obtained with the SI-LAV system. This proposed system is for cost-effective and downscaling analysis, which would be applicable for small hospitals and clinics in remote places with a small number of samples but relatively fast screening would be needed.

Diagnosis and treatment of invasive fungal infections: looking ahead.

Improved standards of care depend on the development of new laboratory diagnostic and imaging procedures and the development of new antifungal compounds. Immunochromatography technologies have led to the development of lateral flow devices for the diagnosis of cryptococcal meningitis and invasive aspergillosis (IA). Similar devices are being developed for the detection of histoplasmosis that meet the requirements for speed (∼15 min assay time) and ease of use for point-of-care diagnostics. The evolution of molecular tools for the detection of fungal pathogens has been slow but the introduction of new nucleic acid amplification techniques appears to be helpful, for example T2Candida. An Aspergillus proximity ligation assay has been developed for a rapid near-patient bedside diagnosis of IA. CT remains the cornerstone for radiological diagnosis of invasive pulmonary fungal infections. MRI of the lungs may be performed to avoid radiation exposure. MRI with T2-weighted turbo-spin-echo sequences exhibits sensitivity and specificity approaching that of CT for the diagnosis of invasive pulmonary aspergillosis. The final part of this review looks at new approaches to drug discovery that have yielded new classes with novel mechanisms of action. There are currently two new classes of antifungal drugs in Phase 2 study for systemic invasive fungal disease and one in Phase 1. These new antifungal drugs show promise in meeting unmet needs with oral and intravenous formulations available and some with decreased potential for drug-drug interactions. Novel mechanisms of action mean these agents are not susceptible to the common resistance mechanisms seen in Candida or Aspergillus. Modification of existing antifungal susceptibility testing techniques may be required to incorporate these new compounds.