ABSTRACT
Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of <10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.
Subject(s)
Lectins , Polysaccharides , Recombinant Proteins , Recombinant Proteins/chemistry , Polysaccharides/chemistry , Polysaccharides/analysis , Lectins/chemistry , Glycosylation , Automation, Laboratory/methodsABSTRACT
We evaluated the DxU 850m Iris Urine Microscopy analyzer as a screening tool for excluding negative urine samples (n = 1337). At a cutoff of 103 colony counts·mL-1, sensitivity was 55.1 %, specificity 68.6 %. The DxU 850m Iris does not offer acceptable prediction of culture-negative urine samples at the tested cutoff.
Subject(s)
Microscopy , Sensitivity and Specificity , Urinalysis , Urine , Humans , Microscopy/methods , Urinalysis/methods , Urinalysis/instrumentation , Urine/microbiology , Urinary Tract Infections/diagnosis , Urinary Tract Infections/microbiology , Automation, Laboratory/methodsABSTRACT
Using sequential immunoassays for the screening of blood donors is well described for viral serology testing but not for the screening of syphilis. In this study, we report the evaluation results and 2-year sequential testing data using two highly sensitive automated serology assays, the Alinity s Syphilis chemiluminescent immunoassay for screening, with all repeatedly reactive samples then tested on the Elecsys Syphilis electrochemiluminescence immunoassay. We screened 1,767,782 blood donor samples between 7 July 2021 and 6 July 2023 and found the Alinity false-positive rate to be low at 0.08% (1,456/1,767,782). The common false-positive rate between the two assays was also low (3.83%, 58/1,514). Concordantly reactive samples were further tested using a Treponema pallidum particle agglutination test, a rapid plasma reagin test, and a fluorescent treponemal antibody absorption test. There were 262/1,376 concordantly reactive Alinity and Elecsys blood donor samples with reactivity on one or more of the confirmatory tests. A total of 26/1,376 donors had a current syphilis infection, 152/1,376 reported a past history of syphilis and had been treated, and 84/1,376 did not report a past history of syphilis. We suggest that future studies could explore the use of sequential immunoassays to aid in the serodiagnosis for syphilis. IMPORTANCE: The serodiagnosis for syphilis usually follows two methodologies-a "traditional" algorithm using a non-treponemal test followed by confirmation using a treponemal test, or a "reverse" algorithm using a treponemal test followed by a non-treponemal test. There are limited reports in the literature of using a modified reverse algorithm (treponemal test followed by a second treponemal test), and to the best of knowledge, there are currently no published articles using two highly sensitive automated immunoassays to aid the serodiagnosis of syphilis. In addition, the Treponema pallidum particle agglutination (TPPA) assay is commonly used as a confirmatory test for the diagnosis of syphilis. With the withdrawal of the TPPA assay from Australia and presumably from the global market also, alternative testing algorithms are now required. This study provides proof of concept for using sequential immunoassays in the diagnosis of syphilis.
Subject(s)
Blood Donors , Syphilis Serodiagnosis , Syphilis , Treponema pallidum , Humans , Syphilis/diagnosis , Syphilis/blood , Immunoassay/methods , Syphilis Serodiagnosis/methods , Treponema pallidum/immunology , Mass Screening/methods , Antibodies, Bacterial/blood , False Positive Reactions , Automation, Laboratory/methods , Sensitivity and Specificity , Female , MaleABSTRACT
Three-dimensional (3D) cell culture creates a more physiologically relevant environment for enhanced drug screening capabilities using microcarriers. An automated 3D system that integrates robotic manipulators, liquid handling systems, sensors, and environment control systems has the capacity to handle multiple samples in parallel, perform repetitive tasks, and provide real-time monitoring and analysis. This chapter describes a potential 3D cell culture drug screening model by combining renal proximal tubule cells as a representative normal cell line with cancer cell lines. This combination is subjected to drug screening to evaluate the drug's efficacy in suppressing cancer cells while minimizing impact on normal cells with the added benefit of having the ability to separate the two cell types by magnetic isolation for high content screens including mass spectrometry-based proteomics. This study presents advancements in 3D cell culture techniques, emphasizing the importance of automation and the potential of microcarriers in drug screening and disease modeling.
Subject(s)
Cell Culture Techniques, Three Dimensional , Humans , Cell Culture Techniques, Three Dimensional/methods , Cell Line, Tumor , Drug Evaluation, Preclinical/methods , Drug Screening Assays, Antitumor/methods , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/metabolism , Cell Culture Techniques/methods , Antineoplastic Agents/pharmacology , Automation , Automation, Laboratory/methods , Neoplasms/pathology , Neoplasms/drug therapyABSTRACT
Quantification of Torquetenovirus (TTV) viremia is becoming important for evaluating the status of the immune system in solid organ transplant recipients, monitoring the appearance of post-transplant complications, and controlling the efficacy of maintenance immunosuppressive therapy. Thus, diagnostic approaches able to scale up TTV quantification are needed. Here, we report on the development and validation of a real-time PCR assay for TTV quantification on the Hologic Panther Fusion® System by utilizing its open-access channel. The manual real-time PCR previously developed in our laboratories was optimized to detect TTV DNA on the Hologic Panther Fusion® System. The assay was validated using clinical samples. The automated TTV assay has a limit of detection of 1.6 log copies per ml of serum. Using 112 samples previously tested via manual real-time PCR, the concordance in TTV detection was 93% between the assays. When the TTV levels were compared, the overall agreement between the methods, as assessed using Passing-Bablok linear regression and Bland-Altman analyses, was excellent. In summary, we validated a highly sensitive and accurate method for the diagnostic use of TTV quantification on a fully automated Hologic Panther Fusion® System. This will greatly improve the turnaround time for TTV testing and better support the laboratory diagnosis of this new viral biomarker.
Subject(s)
DNA Virus Infections , Real-Time Polymerase Chain Reaction , Viral Load , Viremia , Real-Time Polymerase Chain Reaction/methods , Viremia/diagnosis , Viremia/virology , Humans , Viral Load/methods , DNA Virus Infections/diagnosis , DNA Virus Infections/virology , Sensitivity and Specificity , Torque teno virus/genetics , Torque teno virus/isolation & purification , DNA, Viral/genetics , DNA, Viral/blood , Limit of Detection , Reproducibility of Results , Automation, Laboratory/methodsABSTRACT
The ELISA is the most worldwide method for immunoassay. However, the ELISA is losing ground due to low reproducibility of manual experimental processes in both R&D and IVD areas. An automated platform is a good solution, but there are still limitations owning to extremely high cost and requiring large space to set up especially for a small size laboratory. Here, we present a novel all-in-one platform called "VEUS" settable on the laboratory table that offers comprehensive automation of the entire multiplex immunoassay process by exploiting antibody conjugated magnetic particles, quality control and then immunoanalytical reaction, thereby enhancing detection sensitivity and high reproducibility. As a proof of concept, the system exhibits a sensitive LOD of 0.6 and 3.1 pg mL-1 within 1 h run, comparable precision that of molecular diagnostic systems based on PCR method, enabling rapid multiplex diagnosis of Influenza A, Influenza B, and COVID-19 viruses with similar symptoms. Through automation by the all-in-one system, it can be used by novice users, something innovative for immunoassays, relying heavily on user experience. Furthermore, it can contribute to streamline entire immunoassay processes of diverse biomarkers with high reproducibility and convenience in laboratories.
Subject(s)
SARS-CoV-2 , Humans , Immunoassay/methods , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Antibodies, Immobilized/immunology , Antibodies, Immobilized/chemistry , Reproducibility of Results , COVID-19/diagnosis , COVID-19/virology , Enzyme-Linked Immunosorbent Assay/methods , Influenza A virus/immunology , Influenza B virus/immunology , Automation, Laboratory/methods , Limit of DetectionABSTRACT
In the second half of the 20th century, neuroscientists across North America developed automated systems for use in their research laboratories. Their decisions to do so were complex and contingent, partly a result of global reasons, such as the need to increase efficiency and flexibility, and partly a result of local reasons, such as the need to amend perceived biases of earlier research methodologies. Automated methods were advancements but raised several challenges. Transferring a system from one location to another required that certain components of the system be standardized, such as the hardware, software, and programming language. This proved difficult as commercial manufacturers lacked incentives to create standardized products for the few neuroscientists working towards automation. Additionally, investing in automated systems required massive amounts of time, labor, funding, and computer expertise. Moreover, neuroscientists did not agree on the value of automation. My brief history investigates Karl Pribram's decisions to expand his newly created automated system by standardizing equipment, programming, and protocols. Although he was an eminent Stanford neuroscientist with strong institutional support and computer know-how, the development and transfer of his automated behavioral testing system was riddled with challenges. For Pribram and neuroscience more generally, automation was not so automatic.
Subject(s)
Neurosciences , Neurosciences/methods , Neurosciences/history , Neurosciences/instrumentation , History, 20th Century , Automation/methods , Automation/instrumentation , Automation, Laboratory/instrumentation , Automation, Laboratory/methods , Automation, Laboratory/history , North AmericaABSTRACT
BACKGROUND: Viral respiratory Infections pose a health risk, especially to vulnerable patient populations. Effective testing programs can detect and differentiate these infections at an early stage, which is particularly important for high-risk clinical departments. The objective of this study was to develop and validate a multiplex PCR-panel for 16 different respiratory viruses on a fully-automated high-throughput platform. METHODS: Three multiplex-PCR assays were designed to run on the cobas5800/6800/8800 systems, consolidating 16 viral targets: RESP1: SARS-CoV-2, influenza-A/B, RSV; RESP2: hMPV, hBoV, hAdV, rhino-/ENV; RESP3: HPIV-1-4, hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1. Analytic performance was evaluated using digital-PCR based standards and international reference material. Clinical performance was determined by comparing results from clinical samples with reference assays. RESULTS: Analytical sensitivity (i.e. lower limit of detection (LoD), 95 % probability of detection) was determined as follows: SARS-CoV-2: 29.3 IU/ml, influenza-A: 179.9 cp/ml, influenza-B: 333.9 cp/ml and RSV: 283.1 cp/ml. LoDs of other pathogens ranged between 9.4 cp/ml (hCoV-NL63) and 21,419 cp/ml (HPIV-2). Linearity was verified over 4-7 log-steps with pooled standard differentials (SD) ranging between 0.18-0.70ct. Inter-/intra-run variability (precision) was assessed for all targets over 3 days. SDs ranged between 0.13-0.74ct. Positive agreement in clinical samples was 99.4 % and 95 % for SARS-CoV-2 and influenza-A respectively. Other targets were in the 80-100 % range. Negative agreement varied between 96.3-100 %. DISCUSSION: Lab-developed tests are a key factor for effective clinical diagnostics. The multiplex panel presented in this study demonstrated high performance and provides an easily scalable high-throughput solution for respiratory virus testing, e.g. for testing in high-risk patient populations.
Subject(s)
Multiplex Polymerase Chain Reaction , Respiratory Tract Infections , Sensitivity and Specificity , Humans , Multiplex Polymerase Chain Reaction/methods , Respiratory Tract Infections/virology , Respiratory Tract Infections/diagnosis , High-Throughput Screening Assays/methods , Viruses/isolation & purification , Viruses/genetics , Viruses/classification , Virus Diseases/diagnosis , Virus Diseases/virology , Automation, Laboratory/methods , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , COVID-19/diagnosis , COVID-19/virology , Molecular Diagnostic Techniques/methods , Molecular Diagnostic Techniques/standardsABSTRACT
BACKGROUND: Burkholderia pseudomallei, a Gram-negative pathogen, causes melioidosis. Although various clinical laboratory identification methods exist, culture-based techniques lack comprehensive evaluation. Thus, this systematic review and meta-analysis aimed to assess the diagnostic accuracy of culture-based automation and non-automation methods. METHODS: Data were collected via PubMed/MEDLINE, EMBASE, and Scopus using specific search strategies. Selected studies underwent bias assessment using QUADAS-2. Sensitivity and specificity were computed, generating pooled estimates. Heterogeneity was assessed using I2 statistics. RESULTS: The review encompassed 20 studies with 2988 B. pseudomallei samples and 753 non-B. pseudomallei samples. Automation-based methods, particularly with updating databases, exhibited high pooled sensitivity (82.79%; 95% CI 64.44-95.85%) and specificity (99.94%; 95% CI 98.93-100.00%). Subgroup analysis highlighted superior sensitivity for updating-database automation (96.42%, 95% CI 90.01-99.87%) compared to non-updating (3.31%, 95% CI 0.00-10.28%), while specificity remained high at 99.94% (95% CI 98.93-100%). Non-automation methods displayed varying sensitivity and specificity. In-house latex agglutination demonstrated the highest sensitivity (100%; 95% CI 98.49-100%), followed by commercial latex agglutination (99.24%; 95% CI 96.64-100%). However, API 20E had the lowest sensitivity (19.42%; 95% CI 12.94-28.10%). Overall, non-automation tools showed sensitivity of 88.34% (95% CI 77.30-96.25%) and specificity of 90.76% (95% CI 78.45-98.57%). CONCLUSION: The study underscores automation's crucial role in accurately identifying B. pseudomallei, supporting evidence-based melioidosis management decisions. Automation technologies, especially those with updating databases, provide reliable and efficient identification.
Subject(s)
Burkholderia pseudomallei , Melioidosis , Sensitivity and Specificity , Burkholderia pseudomallei/isolation & purification , Melioidosis/diagnosis , Melioidosis/microbiology , Humans , Automation, Laboratory/methods , Bacteriological Techniques/methods , Automation/methodsABSTRACT
Laboratory management automation is essential for achieving interoperability in the domain of experimental research and accelerating scientific discovery. The integration of resources and the sharing of knowledge across organisations enable scientific discoveries to be accelerated by increasing the productivity of laboratories, optimising funding efficiency, and addressing emerging global challenges. This paper presents a novel framework for digitalising and automating the administration of research laboratories through The World Avatar, an all-encompassing dynamic knowledge graph. This Digital Laboratory Framework serves as a flexible tool, enabling users to efficiently leverage data from diverse systems and formats without being confined to a specific software or protocol. Establishing dedicated ontologies and agents and combining them with technologies such as QR codes, RFID tags, and mobile apps, enabled us to develop modular applications that tackle some key challenges related to lab management. Here, we showcase an automated tracking and intervention system for explosive chemicals as well as an easy-to-use mobile application for asset management and information retrieval. Implementing these, we have achieved semantic linking of BIM and BMS data with laboratory inventory and chemical knowledge. Our approach can capture the crucial data points and reduce inventory processing time. All data provenance is recorded following the FAIR principles, ensuring its accessibility and interoperability.
Subject(s)
Automation, Laboratory , Automation, Laboratory/methods , Laboratories , Information Storage and Retrieval/methodsABSTRACT
BACKGROUND: HDV antibody testing is recommended for universal screening and as the first line in an HDV double reflex testing strategy for effectively identifying patients with active infection for therapeutic treatments. OBJECTIVE: The aim of this study is to evaluate the performance of a newly developed ARCHITECT HDV Total Ig (ARCHITECT HDV Ig) prototype assay. STUDY DESIGN: Performance characteristics were determined for the ARCHITECT HDV Ig and a reference test, LIAISON XL Anti-HDV using a well-characterized specimen panel, comprising HDV RNA positive (n = 62) and negative (n = 70) samples, and healthy US blood donors. RESULTS: Healthy US blood donors (n=200) showed 99.5% (199/200, 95%CI=97.65-99.98) specificity with ARCHITECT HDV Ig and 98.5 % (197/200, 95 %CI = 96.10-99.64) with LIAISON Anti-HDV. Among known HDV RNA positive samples, ARCHITECT HDV Ig detected 59/62 demonstrating 95.2 % sensitivity while LIAISON Anti-HDV sensitivity was 90.3 % (56/62). Among 101 HBV positive samples, 70 were reactive in the ARCHITECT test, 59 of which tested positive for HDV RNA for a positive predictive value (PPV) for the presence of HDV RNA was 84.3 %. For LIAISON Anti-HDV, 79 specimens were reactive and 56 contained HDV RNA: PPV for HDV RNA was 70.9 %. Among 70 HDV RNA negative samples, 39 were HBV positive. ARCHITECT HDV Ig negative predictive value (NPV) was 71.8 % and LIAISON Anti-HDV NPV was 41 % for the HBV positive group, respectively. CONCLUSION: When compared to the LIASON Anti-HDV test, the ARCHITECT HDV Ig assay demonstrated enhanced sensitivity and specificity and better NPV and PPV values for HDV RNA status. The ARCHITECT HDV Ig assay represents a promising tool for universal screening of all HBsAg-positive persons.
Subject(s)
Hepatitis Antibodies , Hepatitis D , Hepatitis Delta Virus , High-Throughput Screening Assays , Sensitivity and Specificity , Humans , Hepatitis D/diagnosis , Hepatitis D/immunology , Hepatitis Delta Virus/immunology , Hepatitis Delta Virus/genetics , Hepatitis Delta Virus/isolation & purification , Hepatitis Antibodies/blood , High-Throughput Screening Assays/methods , Serologic Tests/methods , Automation, Laboratory/methods , Blood DonorsABSTRACT
BACKGROUND: In newborns, elevated nucleated red blood cell (NRBC) levels can be associated with enhanced erythropoietic stress and might be predictive for adverse outcome. Also, the presence of NRBC in peripheral blood might lead to erroneous enumeration results of white blood cells in automated hematology analyzers. We aimed to assess the comparability of the Sysmex XN 1000 to manual slide reviews and correlation of NRBC with inflammation markers. METHODS: Specimens of 3397 children under 1 year were compared by automated and microscopic NRBC enumeration. Additionally, potential correlations between NRBC and age and inflammation markers were examined. RESULTS: Overall, there was good correlation (r = 0.97) between automated (range: 0%-3883%) and microscopic enumeration (range: 0%-3694%) of NRBC with high comparability up to a NRBC value of 200% and an increase in the variation between the two methods with increasing NRBC numbers. When 94 samples with ≤ 200% NRBC and ≥ 30% divergence between methods were separately reanalyzed with respect to overlapping cell populations in their scattergrams, Sysmex would have generated unrecognized incorrect automated results in 47 samples, corresponding to 1.4% of total study samples. NRBC counts were negatively correlated to age, but not to inflammation markers. CONCLUSION: Sysmex XN 1000 is highly precise in the enumeration of NRBC in children under 1 year up to counts of 200% and might replace time-intense manual counting in routine diagnostics. In the setting of neonatal and intensive care diagnostics, microscopic control and supervision of scattergrams are highly recommended for any automated NRBC enumeration processes.
Subject(s)
Erythroblasts , Humans , Infant , Erythroblasts/cytology , Infant, Newborn , Erythrocyte Count/methods , Female , Male , Automation, Laboratory/methods , Microscopy/methodsABSTRACT
Potency assays are essential for the development and quality control of biopharmaceutical drugs, but they are often a time limiting factor due to manual handling steps and consequently low analytical throughput. On the other hand, automation of potency assays can be challenging due to their complexity and the use of biological materials. ELISA (enzyme-linked immunosorbent assay) is widely used for potency determination and is a good candidate for automation as all ELISA types depend on the same basic steps: coating, blocking, sample incubation, detection, and signal measurement. Nevertheless, ELISA for relative potency measurements still require drug-specific development and assay validation thereby complicating automation efforts. To simplify potency testing by ELISA, we first developed a manual protocol generally applicable to different drugs and then adapted this protocol for automated measurements. We identified unexpected critical parameters which had to be adapted to transfer the manual ELISA to an automated liquid handling system and we demonstrated that gravimetric sample dilution is unnecessary with the automated protocol. Both manual and automated protocols were validated and compared using multiple biotherapeutics. The automated protocol showed similar or higher precision and accuracy when compared to the manual method.
Subject(s)
Enzyme-Linked Immunosorbent Assay , Enzyme-Linked Immunosorbent Assay/methods , Automation , Immunoglobulin Fragments , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/chemistry , Reproducibility of Results , Humans , Automation, Laboratory/methods , Quality ControlABSTRACT
The virulence of methicillin-resistant Staphylococcus aureus (MRSA) and its potentially fatal outcome necessitate rapid and accurate detection of patients colonized with MRSA in healthcare settings. Using the BD Kiestra Total Lab Automation (TLA) System in conjunction with the MRSA Application (MRSA App), an imaging application that uses artificial intelligence to interpret colorimetric information (mauve-colored colonies) indicative of MRSA pathogen presence on CHROMagar chromogenic media, anterior nares specimens from three sites were evaluated for the presence of mauve-colored colonies. Results obtained with the MRSA App were compared to manual reading of agar plate images by proficient laboratory technologists. Of 1,593 specimens evaluated, 1,545 (96.98%) were concordant between MRSA App and laboratory technologist reading for the detection of MRSA growth [sensitivity 98.15% (95% CI, 96.03, 99.32) and specificity 96.69% (95% CI, 95.55, 97.60)]. This multi-site study is the first evaluation of the MRSA App in conjunction with the BD Kiestra TLA System. Using the MRSA App, our results showed 98.15% sensitivity and 96.69% specificity for the detection of MRSA from anterior nares specimens. The MRSA App, used in conjunction with laboratory automation, provides an opportunity to improve laboratory efficiency by reducing laboratory technologists' labor associated with the review and interpretation of cultures.
Subject(s)
Automation, Laboratory , Bacteriological Techniques , Methicillin-Resistant Staphylococcus aureus , Sensitivity and Specificity , Staphylococcal Infections , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Humans , Staphylococcal Infections/diagnosis , Staphylococcal Infections/microbiology , Automation, Laboratory/methods , Bacteriological Techniques/methods , Automation/methods , Colorimetry/methods , Artificial IntelligenceABSTRACT
In clinical bacteriology laboratories, reading and processing of sterile plates remain a significant part of the routine workload (30%-40% of the plates). Here, an algorithm was developed for bacterial growth detection starting with any type of specimens and using the most common media in bacteriology. The growth prediction performance of the algorithm for automatic processing of sterile plates was evaluated not only at 18-24 h and 48 h but also at earlier timepoints toward the development of an early growth monitoring system. A total of 3,844 plates inoculated with representative clinical specimens were used. The plates were imaged 15 times, and two different microbiologists read the images randomly and independently, creating 99,944 human ground truths. The algorithm was able, at 48 h, to discriminate growth from no growth with a sensitivity of 99.80% (five false-negative [FN] plates out of 3,844) and a specificity of 91.97%. At 24 h, sensitivity and specificity reached 99.08% and 93.37%, respectively. Interestingly, during human truth reading, growth was reported as early as 4 h, while at 6 h, half of the positive plates were already showing some growth. In this context, automated early growth monitoring in case of normally sterile samples is envisioned to provide added value to the microbiologists, enabling them to prioritize reading and to communicate early detection of bacterial growth to the clinicians.
Subject(s)
Artificial Intelligence , Bacteria , Sensitivity and Specificity , Humans , Bacteria/growth & development , Bacteria/isolation & purification , Bacteria/classification , Algorithms , Bacteriological Techniques/methods , Image Processing, Computer-Assisted/methods , Bacterial Infections/diagnosis , Bacterial Infections/microbiology , Bacteriology , Automation, Laboratory/methods , Culture Media/chemistryABSTRACT
The timely identification of microbial pathogens is essential to guide targeted antimicrobial therapy and ultimately, successful treatment of an infection. However, the yield of standard microbiology testing (SMT) is directly related to the duration of antecedent antimicrobial therapy as SMT culture methods are dependent on the recovery of viable organisms, the fastidious nature of certain pathogens, and other pre-analytic factors. In the last decade, metagenomic next-generation sequencing (mNGS) has been successfully utilized as a diagnostic tool for various applications within the clinical laboratory. However, mNGS is resource, time, and labor-intensive-requiring extensive laborious preliminary benchwork, followed by complex bioinformatic analysis. We aimed to address these shortcomings by developing a largely Automated targeted Metagenomic next-generation sequencing (tmNGS) PipeLine for rapId inFectIous disEase Diagnosis (AMPLIFIED) to detect bacteria and fungi directly from clinical specimens. Therefore, AMPLIFIED may serve as an adjunctive approach to complement SMT. This tmNGS pipeline requires less than 1 hour of hands-on time before sequencing and less than 2 hours of total processing time, including bioinformatic analysis. We performed tmNGS on 50 clinical specimens with concomitant cultures to assess feasibility and performance in the hospital laboratory. Of the 50 specimens, 34 (68%) were from true clinical infections. Specimens from cases of true infection were more often tmNGS positive compared to those from the non-infected group (82.4% vs 43.8%, respectively, P = 0.0087). Overall, the clinical sensitivity of AMPLIFIED was 54.6% with 85.7% specificity, equating to 70.6% and 75% negative and positive predictive values, respectively. AMPLIFIED represents a rapid supplementary approach to SMT; the typical time from specimen receipt to identification of potential pathogens by AMPLIFIED is roughly 24 hours which is markedly faster than the days, weeks, and months required to recover bacterial, fungal, and mycobacterial pathogens by culture, respectively. IMPORTANCE: To our knowledge, this represents the first application of an automated sequencing and bioinformatics pipeline in an exclusively pediatric population. Next-generation sequencing is time-consuming, labor-intensive, and requires experienced personnel; perhaps contributing to hesitancy among clinical laboratories to adopt such a test. Here, we report a strong case for use by removing these barriers through near-total automation of our sequencing pipeline.
Subject(s)
Bacteria , Bacterial Infections , Fungi , High-Throughput Nucleotide Sequencing , Metagenomics , Mycoses , Humans , High-Throughput Nucleotide Sequencing/methods , Fungi/genetics , Fungi/isolation & purification , Fungi/classification , Bacteria/genetics , Bacteria/isolation & purification , Bacteria/classification , Bacterial Infections/diagnosis , Bacterial Infections/microbiology , Metagenomics/methods , Mycoses/diagnosis , Mycoses/microbiology , Automation, Laboratory/methods , Sensitivity and Specificity , Molecular Diagnostic Techniques/methods , Time Factors , Computational Biology/methods , Male , Female , Child , Adolescent , Adult , Child, PreschoolABSTRACT
The mpox outbreak, caused by monkeypox virus (MPXV), accelerated the development of molecular diagnostics. In this study, we detail the evaluation of the Research Use Only (RUO) NeuMoDx MPXV assay by multiple European and US sites. The assay was designed and developed by Qiagen for the NeuMoDx Molecular Systems. Primers and probes were tested for specificity and inclusivity in silico. The analytical sensitivity of the assay was determined by testing dilutions of synthetic and genomic MPXV DNA. A total of 296 clinical samples were tested by three sites; the Johns Hopkins University (US), UZ Gent (Belgium, Europe), and Hospital Universitario San Cecilio (Spain, Europe). The analytical sensitivity of the assay was 50 copies/mL for both clades I and II. The assay showed 100% in silico identity for 80 clade I and 99.98% in silico identity for 5,162 clade II genomes. Clade II primers and probes showed 100% in silico specificity; however, identity of at least one of the two sets of clade I primers and probes with variola, cowpox, camelpox, and vaccinia viruses was noticed. The clinical validation showed sensitivity of 99.21% [95% confidence interval (CI): 95.66-99.98%] and specificity of 96.64% (95% CI: 91.62-99.08%) for lesion swab samples. The NeuMoDx MPXV Test shows acceptable analytical and clinical performance. The assay improves the laboratory's workflow as it consolidates nucleic acid extraction, PCR, data analysis, and interpretation and can be interfaced. The Test Strip can differentiate clades I and II, which has important laboratory safety implications. IMPORTANCE: In this manuscript, we provide detailed in silico analysis and clinical evaluation of the assay using a large cohort of clinical samples across three academic centers in Europe and the United States. Because the assay differentiates MPXV clades I and II, this manuscript is timely due to the current need to rule out the regulated clade I by diagnostic clinical laboratories. In December 2023, and due to first report of cases of sexually transmitted clade I infections in the Democratic Republic of the Congo, when generic assays that do not differentiate the clades are used, samples are considered regulated. The assay meets the need of full automation and has a marked positive impact on the laboratory workflow.
Subject(s)
Molecular Diagnostic Techniques , Monkeypox virus , Mpox (monkeypox) , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Humans , Monkeypox virus/genetics , Monkeypox virus/isolation & purification , Monkeypox virus/classification , Real-Time Polymerase Chain Reaction/methods , Mpox (monkeypox)/diagnosis , Mpox (monkeypox)/virology , Molecular Diagnostic Techniques/methods , Europe , United States , Automation, Laboratory/methods , DNA Primers/genetics , BelgiumABSTRACT
After haematology, urinalysis is the most common biological test performed in clinical settings. Hence, simplified workflow and automated analysis of urine elements are of absolute necessities. In the present work, a novel lab-on-chip cartridge (Gravity Sedimentation Cartridge) for the auto analysis of urine elements is developed. The GSC consists of a capillary chamber that uptakes a raw urine sample by capillary force and performs particles and cells enrichment within 5 min through a gravity sedimentation process for the microscopic examination. Centrifugation, which is necessary for enrichment in the conventional method, was circumvented in this approach. The AI100 device (Image based autoanalyzer) captures microscopic images from the cartridge at 40x magnification and uploads them into the cloud. Further, these images were auto-analyzed using an AI-based object detection model, which delivers the reports. These reports were available for expert review on a web-based platform that enables evidence-based tele reporting. A comparative analysis was carried out for various analytical parameters of the data generated through GSC (manual microscopy, tele reporting, and AI model) with the gold standard method. The presented approach makes it a viable product for automated urinalysis in point-of-care and large-scale settings.
Subject(s)
Automation, Laboratory , Lab-On-A-Chip Devices , Urinalysis , Urinalysis/instrumentation , Urinalysis/methods , Humans , Automation, Laboratory/instrumentation , Automation, Laboratory/methods , Artificial IntelligenceABSTRACT
Laboratory capacities are often limited by time-consuming manual repetitive procedures rather than analysis time itself. While modern instruments are typically equipped with an autosampler, sample preparation often follows manual procedures including many labor-intensive, monotonous tasks. Particularly, for a high number of samples, well plates, and low microliter pipetting, manual preparation is error-prone often requiring repeated experiments. Sampling and sample preparation can account for greater analytical variability than instrument analysis. Repetitive tasks such as liquid handling benefit strongly from technological advances and led to the increasing applications of various automated liquid handlers (ALHs). In this review, we discuss the considerations for ALHs in the microliter range and highlight advantages and challenges when transforming from manual to automated workflows. We strongly focused on differences in liquid handling and outlined advantages due to sensor-controlled pipetting. ALHs can substantially improve costs-effectiveness and laboratory capacity. This is a consequence of increased efficiency, and throughput of laboratories while simultaneously raising data quality. Additionally, ALHs can improve safety, documentation of data, and sustainability. While automation requires careful consideration and resource demanding implementation, we believe it offers numerous advantages and can help to transform modern laboratories.