Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components.

The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, we report a pipeline for the rapid design, assembly, and validation of cell-free, paper-based sensors for the detection of the Zika virus RNA genome. By linking isothermal RNA amplification to toehold switch RNA sensors, we detect clinically relevant concentrations of Zika virus sequences and demonstrate specificity against closely related Dengue virus sequences. When coupled with a novel CRISPR/Cas9-based module, our sensors can discriminate between viral strains with single-base resolution. We successfully demonstrate a simple, field-ready sample-processing workflow and detect Zika virus from the plasma of a viremic macaque. Our freeze-dried biomolecular platform resolves important practical limitations to the deployment of molecular diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises. PAPERCLIP.

Ferrobotic swarms enable accessible and adaptable automated viral testing.

Expanding our global testing capacity is critical to preventing and containing pandemics1-9. Accordingly, accessible and adaptable automated platforms that in decentralized settings perform nucleic acid amplification tests resource-efficiently are required10-14. Pooled testing can be extremely efficient if the pooling strategy is based on local viral prevalence15-20; however, it requires automation, small sample volume handling and feedback not available in current bulky, capital-intensive liquid handling technologies21-29. Here we use a swarm of millimetre-sized magnets as mobile robotic agents ('ferrobots') for precise and robust handling of magnetized sample droplets and high-fidelity delivery of flexible workflows based on nucleic acid amplification tests to overcome these limitations. Within a palm-sized printed circuit board-based programmable platform, we demonstrated the myriad of laboratory-equivalent operations involved in pooled testing. These operations were guided by an introduced square matrix pooled testing algorithm to identify the samples from infected patients, while maximizing the testing efficiency. We applied this automated technology for the loop-mediated isothermal amplification and detection of the SARS-CoV-2 virus in clinical samples, in which the test results completely matched those obtained off-chip. This technology is easily manufacturable and distributable, and its adoption for viral testing could lead to a 10-300-fold reduction in reagent costs (depending on the viral prevalence) and three orders of magnitude reduction in instrumentation cost. Therefore, it is a promising solution to expand our testing capacity for pandemic preparedness and to reimagine the automated clinical laboratory of the future.

StratoLAMP: Label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of precipitate.

Multiplex, digital nucleic acid detections have important biomedical applications, but the multiplexity of existing methods is predominantly achieved using fluorescent dyes or probes, making the detection complicated and costly. Here, we present the StratoLAMP for label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of the precipitate byproduct. The StratoLAMP designates two sets of primers with different concentrations to achieve different precipitate yields when amplifying different nucleic acid targets. In the detection, deep learning image analysis is used to stratify the precipitate within each droplet and determine the encapsulated targets for nucleic acid quantification. We investigated the effect of the amplification reagents and process on the precipitate generation and optimized the assay conditions. We then implemented a deep-learning image analysis pipeline for droplet detection, achieving an overall accuracy of 94.3%. In the application, the StratoLAMP successfully achieved the simultaneous quantification of two nucleic acid targets with high accuracy. By eliminating the need for fluorescence, StratoLAMP represents a unique concept toward label-free, multiplex nucleic acid assays and an analytical tool with great cost-effectiveness.

CRISPR-based rapid molecular diagnostic tests for fusion-driven leukemias.

ABSTRACT: Fusion oncogenes can be cancer-defining molecular alterations that are essential for diagnosis and therapy selection.1,2 Rapid and accessible molecular diagnostics for fusion-driven leukemias such as acute promyelocytic leukemia (APL), Philadelphia chromosome-positive acute lymphoblastic leukemia, and chronic myeloid leukemia (CML) are unavailable, creating a barrier to timely diagnosis and effective targeted therapy in many health care settings, including community hospitals and low-resource environments. We developed CRISPR-based RNA-fusion transcript detection assays using SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) for the diagnosis of fusion-driven leukemias. We validated these assays using diagnostic samples from patients with APL and CML from academic centers and dried blood spots from low-resource environments, demonstrating 100% sensitivity and specificity. We identified assay optimizations to enable the use of these tests outside of tertiary cancer centers and clinical laboratories, enhancing the potential impact of this technology. Rapid point-of-care diagnostics can improve outcomes for patients with cancer by expanding access to therapies for highly treatable diseases that would otherwise lead to serious adverse outcomes due to delayed or missed diagnoses.

Practical Guidance for Clinical Microbiology Laboratories: Microbiologic diagnosis of implant-associated infections.

SUMMARYImplant-associated infections (IAIs) pose serious threats to patients and can be associated with significant morbidity and mortality. These infections may be difficult to diagnose due, in part, to biofilm formation on device surfaces, and because even when microbes are found, their clinical significance may be unclear. Despite recent advances in laboratory testing, IAIs remain a diagnostic challenge. From a therapeutic standpoint, many IAIs currently require device removal and prolonged courses of antimicrobial therapy to effect a cure. Therefore, making an accurate diagnosis, defining both the presence of infection and the involved microorganisms, is paramount. The sensitivity of standard microbial culture for IAI diagnosis varies depending on the type of IAI, the specimen analyzed, and the culture technique(s) used. Although IAI-specific culture-based diagnostics have been described, the challenge of culture-negative IAIs remains. Given this, molecular assays, including both nucleic acid amplification tests and next-generation sequencing-based assays, have been used. In this review, an overview of these challenging infections is presented, as well as an approach to their diagnosis from a microbiologic perspective.

Nucleic acid hybridization-based detection of pathogenic RNA using microscale thermophoresis.

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.

Variant classification changes over time in the clinical molecular diagnostic laboratory setting.

BACKGROUND: Variant classification in the setting of germline genetic testing is necessary for patients and their families to receive proper care. Variants are classified as pathogenic (P), likely pathogenic (LP), uncertain significance (VUS), likely benign (LB) and benign (B) using the standards and guidelines recommended by the American College of Medical Genetics and the Association for Molecular Pathology, with modifications for specific genes. As the literature continues to rapidly expand, and evidence continues to accumulate, prior classifications can be updated accordingly. In this study, we aim to characterise variant reclassifications in Ontario. METHODS: DNA samples from patients seen at hereditary cancer clinics in Ontario from January 2012 to April 2022 were submitted for testing. Patients met provincial eligibility criteria for testing for hereditary cancer syndromes or polycystic kidney disease. Reclassification events were determined to be within their broader category of significance (B to LB or vice versa, or P to LP or vice versa) or outside of their broader category as significance (ie, significant reclassifications from B/LB or VUS or P/LP, from P/LP to VUS or B/LB, or from VUS to any other category). RESULTS: Of the 8075 unique variants included in this study, 23.7% (1912) of variants were reassessed, and 7.2% (578) of variants were reclassified. Of these, 351 (60.7%) variants were reclassified outside of their broader category of significance. Overall, the final classification was significantly different for 336 (58.1%) variants. Importantly, most reclassified variants were downgraded to a more benign classification (n=245; 72.9%). Of note, most reclassified VUS was downgraded to B/LB (n=233; 84.7%). CONCLUSIONS: The likelihood for reclassification of variants on reassessment is high. Most reclassified variants were downgraded to a more benign classification. Our findings highlight the importance of periodic variant reassessment to ensure timely and appropriate care for patients and their families.

Clinical Impact of Multiplex Molecular Diagnostic Testing in Children With Acute Gastroenteritis Presenting to an Emergency Department: A Multicenter Prospective Study.

BACKGROUND: Multiplex molecular diagnostic panels have greatly enhanced detection of gastrointestinal pathogens. However, data on the impact of these tests on clinical and patient-centered outcomes are limited. METHODS: We conducted a prospective, multicenter, stepped-wedge trial to determine the impact of multiplex molecular testing at 5 academic children's hospitals on children presenting to the emergency department with acute gastroenteritis. Caregivers were interviewed on enrollment and 7-10 days after enrollment to determine symptoms, risk factors, subsequent medical visits, and impact on family members. During the pre-intervention period, diagnostic testing was performed at the clinician's discretion . During the intervention period, multiplex molecular testing was performed on all children, with results available to clinicians. The primary outcome was return visits to a healthcare provider within 10 days of enrollment. RESULTS: Potential pathogens were identified by clinician-ordered tests in 19 of 571 (3.3%) in the pre-intervention period compared with 434 of 586 (74%) in the intervention period; clinically relevant pathogens were detected in 2.1% and 15%, respectively. In the multivariate model, the intervention was associated with a 21% reduction in the odds of any return visit (odds ratio, 0.79; 95% confidence interval, .70-.90) after adjusting for potential confounders. Appropriate treatment was prescribed in 11.3% compared with 19.6% during the intervention period (P = .22). CONCLUSIONS: Routine molecular multiplex testing for all children who presented to the ED with acute gastroenteritis detected more clinically relevant pathogens and led to a 21% decrease in return visits. Additional research is needed to define patients most likely to benefit from testing. Clinical Trials Registration. NCT02248285.

New Manual Quantitative Polymerase Chain Reaction Assay Validated on Tongue Swabs Collected and Processed in Uganda Shows Sensitivity That Rivals Sputum-based Molecular Tuberculosis Diagnostics.

BACKGROUND: Sputum-based testing is a barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce sputum, and sputum processing increases assay complexity and cost. Tongue swabs are emerging as an alternative to sputum, but performance limits are uncertain. METHODS: From June 2022 to July 2023, we enrolled 397 consecutive adults with cough >2 weeks at 2 health centers in Kampala, Uganda. We collected demographic and clinical information, sputum for TB testing (Xpert MTB/RIF Ultra and 2 liquid cultures), and tongue swabs for same-day quantitative polymerase chain reaction (qPCR) testing. We evaluated tongue swab qPCR diagnostic accuracy versus sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared 2 swab types (Copan FLOQSWAB and Steripack spun polyester). RESULTS: Among 397 participants, 43.1% were female, median age was 33 years, 23.5% were diagnosed with human immunodeficiency virus, and 32.0% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% (95% confidence interval [CI]: 96.2-99.1) of participants. Tongue swab qPCR sensitivity was 92.6% (95% CI: 86.5 to 96.0) and specificity was 99.1% (95% CI: 96.9 to 99.8) versus microbiological reference standard. A single tongue swab recovered a 7-log range of TB copies, with a decreasing recovery trend among 4 serial swabs. Swab types performed equivalently. CONCLUSIONS: Tongue swabs are a promising alternative to sputum for molecular diagnosis of TB, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics.

The Infectious Diseases Society of America Guidelines on the Diagnosis of COVID-19: Molecular Diagnostic Testing (December 2023).

Accurate molecular diagnostic tests are necessary for confirming a diagnosis of coronavirus disease 2019 (COVID-19) and for identifying asymptomatic carriage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The number of available SARS-CoV-2 nucleic acid detection tests continues to increase as does the COVID-19 diagnostic literature. Thus, the Infectious Diseases Society of America (IDSA) developed an evidence-based diagnostic guideline to assist clinicians, clinical laboratorians, patients, and policymakers in decisions related to the optimal use of SARS-CoV-2 nucleic acid amplification tests. In addition, we provide a conceptual framework for understanding molecular diagnostic test performance, discuss nuances of test result interpretation in a variety of practice settings, and highlight important unmet research needs related to COVID-19 diagnostic testing. IDSA convened a multidisciplinary panel of infectious diseases clinicians, clinical microbiologists, and experts in systematic literature review to identify and prioritize clinical questions and outcomes related to the use of SARS-CoV-2 molecular diagnostics. Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology was used to assess the certainty of evidence and make testing recommendations. The panel agreed on 12 diagnostic recommendations. Access to accurate SARS-CoV-2 nucleic acid testing is critical for patient care, hospital infection prevention, and the public health response to COVID-19 infection. Information on the clinical performance of available tests continues to grow, but the quality of evidence of the current literature to support this updated molecular diagnostic guideline remains moderate to very low. Recognizing these limitations, the IDSA panel weighed available diagnostic evidence and recommends nucleic acid testing for all symptomatic individuals suspected of having COVID-19. In addition, testing is suggested for asymptomatic individuals with known or suspected contact with a COVID-19 case when the results will impact isolation/quarantine/personal protective equipment (PPE) usage decisions. Evidence in support of rapid testing and testing of upper respiratory specimens other than nasopharyngeal swabs, which offer logistical advantages, is sufficient to warrant conditional recommendations in favor of these approaches.

Loop-Mediated Isothermal Amplification Assay to Detect Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 ng of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.

Low-cost CRISPR diagnostics for resource-limited settings.

Next-generation sequencing (NGS) has identified disease hallmarks and catalogued a vast reservoir of genetic information from humans and other species. Precise nucleotide-interrogation properties of clustered regularly interspaced short palindromic repeats (CRISPR) proteins have been harnessed to rapidly identify DNA-RNA signatures for diverse applications, bypassing the cost and turnaround times associated with diagnostic NGS.

Enhancing Loop-Mediated Isothermal Amplification through Nonpowered Nanoelectric Preconcentration.

The global threat posed by the COVID-19 pandemic has catalyzed the development of point-of-care (POC) molecular diagnostics. While loop-mediated isothermal amplification (LAMP) stands out as a promising technique among FDA-approved methods, it is occasionally susceptible to a high risk of false positives due to nonspecific amplification of a primer dimer. In this work, we report an enhancing LAMP technique in terms of assay sensitivity and reliability through streamlined integration with a nonpowered nanoelectric preconcentration (NPP). The NPP, serving as a sample preparation tool, enriched the virus concentration in samples prior to the subsequent LAMP assay. This enrichment enabled not only to achieve more sensitive assay but also to shorten the assay time for all tested clinical samples by ∼10 min compared to the conventional LAMP. The shortened assay time suppresses the occurrence of nonspecific amplification by not providing the necessary incubation time, effectively suppressing misidentification by false positives. Utilizing this technique, we also developed a prototype of the POC NPP-LAMP kit. This kit offers a streamlined diagnostic process for nontrained individuals, from the sample enrichment, transfer of the enriched sample to LAMP assays, which facilitates on-site/on-demand diagnosis of SARS-CoV-2. This development holds the potential to contribute toward preventing not only the current outbreak but also future occurrences of pandemic viruses.

Rapid, Portable, and Electricity-free Sample Extraction Method for Enhanced Molecular Diagnostics in Resource-Limited Settings.

The COVID-19 pandemic has highlighted the need for rapid and reliable diagnostics that are accessible in resource-limited settings. To address this pressing issue, we have developed a rapid, portable, and electricity-free method for extracting nucleic acids from respiratory swabs (i.e. nasal, nasopharyngeal and buccal swabs), successfully demonstrating its effectiveness for the detection of SARS-CoV-2 in residual clinical specimens. Unlike traditional approaches, our solution eliminates the need for micropipettes or electrical equipment, making it user-friendly and requiring little to no training. Our method builds upon the principles of magnetic bead extraction and revolves around a low-cost plastic magnetic lid, called SmartLid, in combination with a simple disposable kit containing all required reagents conveniently prealiquoted. Here, we clinically validated the SmartLid sample preparation method in comparison to the gold standard QIAamp Viral RNA Mini Kit from QIAGEN, using 406 clinical isolates, including 161 SARS-CoV-2 positives, using the SARS-CoV-2 RT-qPCR assays developed by the US Centers for Disease Control and Prevention (CDC). The SmartLid method showed an overall sensitivity of 95.03% (95% CI: 90.44-97.83%) and a specificity of 99.59% (95% CI: 97.76-99.99%), with a positive agreement of 97.79% (95% CI: 95.84-98.98%) when compared to QIAGEN's column-based extraction method. There are clear benefits to using the SmartLid sample preparation kit: it enables swift extraction of viral nucleic acids, taking less than 5 min, without sacrificing significant accuracy when compared to more expensive and time-consuming alternatives currently available on the market. Moreover, its simplicity makes it particularly well-suited for the point-of-care where rapid results and portability are crucial. By providing an efficient and accessible means of nucleic acid extraction, our approach aims to introduce a step-change in diagnostic capabilities for resource-limited settings.

From Lab to Home: Ultrasensitive Rapid Detection of SARS-CoV-2 with a Cascade CRISPR/Cas13a-Cas12a System Based Lateral Flow Assay.

Currently, CRISPR/Cas-based molecular diagnostic techniques usually rely on the introduction of nucleic acid amplification to improve their sensitivity, which is usually more time-consuming, susceptible to aerosol contamination, and therefore not suitable for at-home molecular testing. In this research, we developed an advanced CRISPR/Cas13a-Cas12a-based lateral flow assay that facilitated the ultrasensitive and rapid detection of SARS-CoV-2 RNA directly from samples, without the need for nucleic acid amplification. This method was called CRISPR LFA enabling at-home RNA testing (CLEAR). CLEAR used a novel cascade mechanism with specially designed probes that fold into hairpin structures, enabling visual detection of SARS-CoV-2 sequences down to 1 aM sensitivity levels. More importantly, CLEAR had a positive coincidence rate of 100% and a negative coincidence rate of 100% for clinical nasopharyngeal swabs from 16 patients. CLEAR was particularly suitable for at-home molecular testing, providing a low-cost, user-friendly solution that can efficiently distinguish between different SARS-CoV-2 variants. CLEAR overcame the common limitations of high sensitivity and potential contamination associated with traditional PCR-based systems, making it a promising tool for widespread public health application, especially in environments with limited access to laboratory resources.

Mini-Program Enabled IoT Intelligent Molecular Diagnostic Device for Co-Detection and Spatiotemporal Mapping of Infectious Disease Pathogens.

Effective detection of infectious pathogens is crucial for disease prevention and control. We present an innovative Internet of Things (IoT) molecular diagnostic device featuring a WeChat mini-program for simultaneous detection and spatiotemporal mapping of respiratory pathogens. Leveraging social software's widespread usage, our device integrates seamlessly with WeChat, eliminating the need for app downloads and installations. Through a comprehensive detection system, including a user-friendly mini-program, a portable Point-of-Care fluorescence detector, and a diagnostic information management platform (EzDx Cloud), we demonstrate high sensitivity and specificity in detecting common respiratory viruses. Our SARS-CoV-2/H1N1 combo test kit, developed using a novel one-tube/one-step loop-mediated isothermal amplification-CRISPR method, shows remarkable performance. We address challenges in at-home nucleic acid testing by providing a cost-effective solution capable of detecting multiple pathogens simultaneously. Our system's versatility accommodates various assays operating at different temperatures and fluorescence intensities, offering significant advantages over traditional methods. Moreover, integration with EzDx Cloud facilitates disease monitoring and early warning systems, enhancing public health management. This study highlights the potential of our IoT molecular diagnostic device in revolutionizing infectious disease detection and control, with wide-ranging applications in both human and animal population.

A rapid on-site loop-mediated isothermal amplification technology as an early warning system for the detection of Shiga toxin-producing Escherichia coli in water.

Shiga toxin-producing Escherichia coli (STEC) is an important waterborne pathogen capable of causing serious gastrointestinal infections with potentially fatal complications, including haemolytic-uremic syndrome. All STEC serogroups harbour genes that encode at least one Shiga toxin (stx1 and/or stx2), which constitute the primary virulence factors of STEC. Loop-mediated isothermal amplification (LAMP) enables rapid real-time pathogen detection with a high degree of specificity and sensitivity. The aim of this study was to develop and validate an on-site portable diagnostics workstation employing LAMP technology to permit rapid real-time STEC detection in environmental water samples. Water samples (n=28) were collected from groundwater wells (n=13), rivers (n=12), a turlough (n=2) and an agricultural drain (n=1) from the Corrib catchment in Galway. Water samples (100 ml) were passed through a 0.22 µm filter, and buffer was added to elute captured cells. Following filtration, eluates were tested directly using LAMP assays targeting stx1, stx2 and E. coli phoA genes. The portable diagnostics workstation was used in field studies to demonstrate the on-site testing capabilities of the instrument. Real-time PCR assays targeting stx1 and stx2 genes were used to confirm the results. The limit of detection for stx1, stx2 and phoA LAMP assays were 2, 2 and 6 copies, respectively. Overall, stx1, stx2 and phoA genes were detected by LAMP in 15/28 (53.6 %), 9/28 (32.2 %) and 24/28 (85.7 %) samples, respectively. For confirmation, the LAMP results for stx1 and stx2 correlated perfectly (100 %) with those obtained using PCR. The portable diagnostics workstation exhibited high sensitivity throughout the on-site operation, and the average time from sample collection to final result was 40 min. We describe a simple, transferable and efficient diagnostic technology for on-site molecular analysis of various water sources. This method allows on-site testing of drinking water, enabling evidence-based decision-making by public health and water management authorities.

Application of LAMP coupled with NALF for precise detection of mycoplasma pneumoniae.

Mycoplasma pneumoniae (MP),as the most commonly infected respiratory pathogen in community-acquired pneumonia in preschool children,has becoming a prominent factor affecting children's respiratory health.Currently, there is a lack of easy, rapid, and accurate laboratory testing program for MP infection, which causes comparatively difficulty for clinical diagnostic.Here,we utilize loop-mediated isothermal amplification (LAMP) to amplify and characterize the P1 gene of MP, combined with nucleic acid lateral flow (NALF) for fast and visuallized detection of MP.Furthermore, we evaluated and analyzed the sensitivity, specificity and methodological consistency of the method.The results showed that the limit of detection(LoD) of MP-LAMP-NALF assay was down to 100 copys per reaction and there was no cross-reactivity with other pathogens infected the respiratory system. The concordance rate between MP-LAMP-NALF assay with quantitative real-time PCR was 94.3 %,which exhibiting excellent testing performance.We make superior the turnaround time of the MP-LAMP-NALF assay, which takes only about 50 min. In addition, there is no need for precision instruments and no restriction on the laboratory site.Collectively, LAMP-NALF assay targeting the P1 gene for Mycoplasma pneumoniae detection was a easy, precise and visual test which could be widely applied in outpatient and emergency departments or primary hospitals.When further optimized, it could be used as "point-of-care testing" of pathogens or multiple testing for pathogens.

A simple and sensitive test for Candida auris colonization, surveillance, and infection control suitable for near patient use.

Candida auris is a multidrug-resistant fungal pathogen with a propensity to colonize humans and persist on environmental surfaces. C. auris invasive fungal disease is being increasingly identified in acute and long-term care settings. We have developed a prototype cartridge-based C. auris surveillance assay (CaurisSurV cartridge; "research use only") that includes integrated sample processing and nucleic acid amplification to detect C. auris from surveillance skin swabs in the GeneXpert instrument and is designed for point-of-care use. The assay limit of detection (LoD) in the skin swab matrix was 10.5 and 14.8 CFU/mL for non-aggregative (AR0388) and aggregative (AR0382) strains of C. auris, respectively. All five known clades of C. auris were detected at 2-3-5× (31.5-52.5 CFU/mL) the LoD. The assay was validated using a total of 85 clinical swab samples banked at two different institutions (University of California Los Angeles, CA and Wadsworth Center, NY). Compared to culture, sensitivity was 96.8% (30/31) and 100% (10/10) in the UCLA and Wadsworth cohorts, respectively, providing a combined sensitivity of 97.5% (40/41), and compared to PCR, the combined sensitivity was 92% (46/50). Specificity was 100% with both clinical (C. auris negative matrix, N = 31) and analytical (non-C. auris strains, N = 32) samples. An additional blinded study with N = 60 samples from Wadsworth Center, NY yielded 97% (29/30) sensitivity and 100% (28/28) specificity. We have developed a completely integrated, sensitive, specific, and 58-min prototype test, which can be used for routine surveillance of C. auris and might help prevent colonization and outbreaks in acute and chronic healthcare settings. IMPORTANCE: This study has the potential to offer a better solution to healthcare providers at hospitals and long-term care facilities in their ongoing efforts for effective and timely control of Candida auris infection and hence quicker response for any potential future outbreaks.

Comparative evaluation of the STANDARD M10 and Xpert C. difficile assays for detection of toxigenic Clostridioides difficile in stool specimens.

This study compared the performance of two commercial molecular assays, the STANDARD M10 Clostridioides difficile assay (M10) and the Xpert C. difficile assay (Xpert), for detecting toxigenic C. difficile in stool specimens. A total of 487 consecutive stool specimens submitted for routine C. difficile testing between June and November 2023 were included. Following routine testing using C. DIFF QUIK CHEK COMPLETE (QCC), M10 and Xpert were tested in parallel, alongside toxigenic culture (reference standard). Additionally, two-step algorithms, using QCC on the first step and either M10 or Xpert on the second step, were assessed. Both M10 and Xpert demonstrated a sensitivity and negative predictive value (NPV) of 100%. M10 exhibited significantly higher specificity and positive predictive value (PPV; 91.9% and 64.2%, respectively) than Xpert (90.3% and 59.8%, respectively). Both two-step algorithms showed a sensitivity and NPV of 98.4% and 99.8%, respectively. The specificity and PPV of the two-step algorithm using M10 (95.2% and 75.0%, respectively) were slightly higher than those of the one using Xpert (94.8% and 73.2%, respectively), without statistical significance. Receiver operating characteristic curve analysis, assessing the predictive ability of cycle threshold (Ct) values for the detection of free toxin, exhibited an area under the curve of 0.825 for M10 and 0.843 for Xpert. This indicates the utility of Ct values as predictors for the detection of free toxin in both assays. In conclusion, M10 proves to be an effective diagnostic tool with performance comparable to Xpert, whether utilized independently or as part of a two-step algorithm.