ABSTRACT
ABSTRACTImproved sanitation, increased access to health care, and advances in preventive and clinical medicine have reduced the mortality and morbidity rates of several infectious diseases. However, recent outbreaks of several emerging infectious diseases (EIDs) have caused substantial mortality and morbidity, and the frequency of these outbreaks is likely to increase due to pathogen, environmental, and population effects driven by climate change. Extreme or persistent changes in temperature, precipitation, humidity, and air pollution associated with climate change can, for example, expand the size of EID reservoirs, increase host-pathogen and cross-species host contacts to promote transmission or spillover events, and degrade the overall health of susceptible host populations leading to new EID outbreaks. It is therefore vital to establish global strategies to track and model potential responses of candidate EIDs to project their future behaviour and guide research efforts on early detection and diagnosis technologies and vaccine development efforts for these targets. Multi-disciplinary collaborations are demanding to develop effective inter-continental surveillance and modelling platforms that employ artificial intelligence to mitigate climate change effects on EID outbreaks. In this review, we discuss how climate change has increased the risk of EIDs and describe novel approaches to improve surveillance of emerging pathogens that pose the risk for EID outbreaks, new and existing measures that could be used to contain or reduce the risk of future EID outbreaks, and new methods to improve EID tracking during further outbreaks to limit disease transmission.
Subject(s)
Climate Change , Communicable Diseases, Emerging , Humans , Communicable Diseases, Emerging/epidemiology , Communicable Diseases, Emerging/prevention & control , Communicable Diseases, Emerging/transmission , Animals , Disease Outbreaks/prevention & controlABSTRACT
Extracellular vesicles (EVs) secreted by all cell types are involved in the cell-to-cell transfer of regulatory factors that influence cell and tissue phenotypes in normal and diseased tissues. EVs are thus a rich source of biomarker targets for assays that analyze blood and urinary EVs for disease diagnosis. Sensitive biomarker detection in EVs derived from specific cell populations is a key major hurdle when analyzing complex biological samples, but innovative approaches surveyed in this Perspective can streamline EV isolation and enhance the sensitivity of EV detection procedures required for clinical application of EV-based diagnostics and therapeutics, including nanotechnology and microfluidics, to achieve EV characterizations. Finally, this Perspective also outlines opportunities and challenges remaining for clinical translation of EV-based assays.
Subject(s)
Extracellular Vesicles , Biomarkers/metabolism , Extracellular Vesicles/metabolism , Phenotype , Nanotechnology , Biological TransportABSTRACT
Rationale: Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial (NTM) pulmonary disease (PD), which exhibits increasing global incidence. Current microbiologic methods routinely used in clinical practice lack sensitivity and have long latencies, leading to delays in diagnosis and treatment initiation and evaluation. A clustered regularly interspaced short palindromic repeats (CRISPR)-based assay that measures MAC cell-free DNA (cfDNA) concentrations in serum could provide a rapid means to detect MAC infection and monitor response to antimicrobial treatment. Objectives: To develop and optimize a CRISPR MAC assay for MAC infection detection and to evaluate its diagnostic and prognostic performance in two MAC disease cohorts. Methods: MAC cfDNA serum concentrations were measured in individuals with diagnoses of MAC disease or who had bronchiectasis or chronic obstructive pulmonary disease diagnoses without histories of NTM PD or NTM-positive sputum cultures. Diagnostic performance was analyzed using pretreatment serum from two cohorts. Serum MAC cfDNA changes during MAC PD treatment were evaluated in a subset of patients with MAC PD who received macrolide-based multidrug regimens. Measurements and Main Results: The CRISPR MAC assay detected MAC cfDNA in MAC PD with 97.6% (91.6-99.7%) sensitivity and 97.6% (91.5-99.7%) specificity overall. Serum MAC cfDNA concentrations markedly decreased after MAC-directed treatment initiation in patients with MAC PD who demonstrated MAC culture conversion. Conclusions: This study provides preliminary evidence for the utility of a serum-based CRISPR MAC assay to rapidly detect MAC infection and monitor the response to treatment.
Subject(s)
Cell-Free Nucleic Acids , Mycobacterium avium Complex , Mycobacterium avium-intracellulare Infection , Humans , Mycobacterium avium-intracellulare Infection/diagnosis , Mycobacterium avium-intracellulare Infection/blood , Mycobacterium avium-intracellulare Infection/drug therapy , Female , Male , Cell-Free Nucleic Acids/blood , Mycobacterium avium Complex/genetics , Mycobacterium avium Complex/isolation & purification , Aged , Middle Aged , DNA, Bacterial/blood , DNA, Bacterial/analysis , Sensitivity and Specificity , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Cohort Studies , Anti-Bacterial Agents/therapeutic useABSTRACT
BACKGROUND: Novel approaches that allow early diagnosis and treatment monitoring of both human immunodeficiency virus-1 (HIV-1) and tuberculosis disease (TB) are essential to improve patient outcomes. METHODS: We developed and validated an immuno-affinity liquid chromatography-tandem mass spectrometry (ILM) assay that simultaneously quantifies single peptides derived from HIV-1 p24 and Mycobacterium tuberculosis (Mtb) 10-kDa culture filtrate protein (CFP10) in trypsin-digested serum derived from cryopreserved serum archives of cohorts of adults and children with/without HIV and TB. RESULTS: ILM p24 and CFP10 results demonstrated good intra-laboratory precision and accuracy, with recovery values of 96.7% to 104.6% and 88.2% to 111.0%, total within-laboratory precision (CV) values of 5.68% to 13.25% and 10.36% to 14.92%, and good linearity (r2 > 0.99) from 1.0 to 256.0â pmol/L and 0.016 to 16.000â pmol/L, respectively. In cohorts of adults (n = 34) and children (n = 17) with HIV and/or TB, ILM detected p24 and CFP10 demonstrated 85.7% to 88.9% and 88.9% to 100.0% diagnostic sensitivity for HIV-1 and TB, with 100% specificity for both, and detected HIV-1 infection earlier than 3 commercial p24 antigen/antibody immunoassays. Finally, p24 and CFP10 values measured in longitudinal serum samples from children with HIV-1 and TB distinguished individuals who responded to TB treatment from those who failed to respond or were untreated, and who developed TB immune reconstitution inflammatory syndrome. CONCLUSIONS: Simultaneous ILM evaluation of p24 and CFP10 results may allow for early TB and HIV detection and provide valuable information on treatment response to facilitate integration of TB and HIV diagnosis and management.
Subject(s)
HIV Infections , HIV-1 , Mycobacterium tuberculosis , Adult , Child , Humans , Tandem Mass Spectrometry , HIV Infections/diagnosis , Peptides , Chromatography, Liquid , Sensitivity and SpecificityABSTRACT
Extensive effort has been devoted to the discovery, development, and validation of biomarkers for early disease diagnosis and prognosis as well as rapid evaluation of the response to therapeutic interventions. Genomic and transcriptomic profiling are well-established means to identify disease-associated biomarkers. However, analysis of disease-associated peptidomes can also identify novel peptide biomarkers or signatures that provide sensitive and specific diagnostic and prognostic information for specific malignant, chronic, and infectious diseases. Growing evidence also suggests that peptidomic changes in liquid biopsies may more effectively detect changes in disease pathophysiology than other molecular methods. Knowledge gained from peptide-based diagnostic, therapeutic, and imaging approaches has led to promising new theranostic applications that can increase their bioavailability in target tissues at reduced doses to decrease side effects and improve treatment responses. However, despite major advances, multiple factors can still affect the utility of peptidomic data. This review summarizes several remaining challenges that affect peptide biomarker discovery and their use as diagnostics, with a focus on technological advances that can improve the detection, identification, and monitoring of peptide biomarkers for personalized medicine.
ABSTRACT
Tuberculosis (TB) remains a major underdiagnosed public health threat worldwide, being responsible for more than 10 million cases and one million deaths annually. TB diagnosis has become more rapid with the development and adoption of molecular tests, but remains challenging with traditional TB diagnosis, but there has not been a critical review of this area. Here, we systematically review these approaches to assess their diagnostic potential and issues with the development and clinical evaluation of proposed CRISPR-based TB assays. Based on these observations, we propose constructive suggestions to improve sample pretreatment, method development, clinical validation, and accessibility of these assays to streamline future assay development and validation studies.
Subject(s)
Biological Assay , Tuberculosis , Humans , Public Health , Tuberculosis/diagnosis , Tuberculosis/geneticsABSTRACT
Numerous groups have employed the special properties of CRISPR/Cas systems to develop platforms that have broad potential applications for sensitive and specific detection of nucleic acid (NA) targets. However, few of these approaches have progressed to commercial or clinical applications. This review summarizes the properties of known CRISPR/Cas systems and their applications, challenges associated with the development of such assays, and opportunities to improve their performance or address unmet assay needs using nano-/micro-technology platforms. These include rapid and efficient sample preparation, integrated single-tube, amplification-free, quantifiable, multiplex, and non-NA assays. Finally, this review discusses the current outlook for such assays, including remaining barriers for clinical or point-of-care applications and their commercial development.
Subject(s)
CRISPR-Cas Systems , Nucleic Acids , CRISPR-Cas Systems/genetics , Specimen HandlingABSTRACT
CRISPR-based assays can be adopted as ultrasensitive molecular diagnostics in resource-limited settings, but point-of-care applications must address additional requirements. Here, we discuss the major obstacles for developing these assays and offer insights into how to surmount them.
ABSTRACT
Integrins expressed on extracellular vesicles (EVs) secreted by various cancers are reported to mediate the organotropism of these EVs. Our previous experiment found that pancreatic tissue of mice with severe cases of acute pancreatitis (SAP) overexpresses several integrins and that serum EVs of these mice (SAP-EVs) can mediate acute lung injury (ALI). It is unclear if SAP-EV express integrins that can promote their accumulation in the lung to promote ALI. Here, we report that SAP-EV overexpress several integrins and that preincubation of SAP-EV with the integrin antagonist peptide HYD-1 markedly attenuates their pulmonary inflammation and disrupt the pulmonary microvascular endothelial cell (PMVEC) barrier. Further, we report that injecting SAP mice with EVs engineered to overexpress two of these integrins (ITGAM and ITGB2) can attenuate the pulmonary accumulation of pancreas-derived EVs and similarly decrease pulmonary inflammation and disruption of the endothelial cell barrier. Based on these findings, we propose that pancreatic EVs can mediate ALI in SAP patients and that this injury response could be attenuated by administering EVs that overexpress ITGAM and/or ITGB2, which is worthy of further study due to the lack of effective therapies for SAP-induced ALI.
Subject(s)
Acute Lung Injury , Pancreatitis , Mice , Animals , Acute Disease , Tumor Necrosis Factor-alpha , Lung , IntegrinsABSTRACT
Neurologic manifestations are among the most frequently reported complications of COVID-19. However, given the paucity of tissue samples and the highly infectious nature of the etiologic agent of COVID-19, we have limited information to understand the neuropathogenesis of COVID-19. Therefore, to better understand the impact of COVID-19 on the brain, we used mass-spectrometry-based proteomics with a data-independent acquisition mode to investigate cerebrospinal fluid (CSF) proteins collected from two different nonhuman primates, Rhesus Macaque and African Green Monkeys, for the neurologic effects of the infection. These monkeys exhibited minimal to mild pulmonary pathology but moderate to severe central nervous system (CNS) pathology. Our results indicated that CSF proteome changes after infection resolution corresponded with bronchial virus abundance during early infection and revealed substantial differences between the infected nonhuman primates and their age-matched uninfected controls, suggesting these differences could reflect altered secretion of CNS factors in response to SARS-CoV-2-induced neuropathology. We also observed the infected animals exhibited highly scattered data distributions compared to their corresponding controls indicating the heterogeneity of the CSF proteome change and the host response to the viral infection. Dysregulated CSF proteins were preferentially enriched in functional pathways associated with progressive neurodegenerative disorders, hemostasis, and innate immune responses that could influence neuroinflammatory responses following COVID-19. Mapping these dysregulated proteins to the Human Brain Protein Atlas found that they tended to be enriched in brain regions that exhibit more frequent injury following COVID-19. It, therefore, appears reasonable to speculate that such CSF protein changes could serve as signatures for neurologic injury, identify important regulatory pathways in this process, and potentially reveal therapeutic targets to prevent or attenuate the development of neurologic injuries following COVID-19.
Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Humans , Chlorocebus aethiops , Cerebrospinal Fluid Proteins , Proteome , Macaca mulattaABSTRACT
Interferon-gamma release assays (IGRAs) that measure pathogen-specific T-cell response rates can provide a more reliable estimate of protection than specific antibody levels but have limited potential for widespread use due to their workflow, personnel, and instrumentation demands. The major vaccines for SARS-CoV-2 have demonstrated substantial efficacy against all of its current variants, but approaches are needed to determine how these vaccines will perform against future variants, as they arise, to inform vaccine and public health policies. Here we describe a rapid, sensitive, nanolayer polylysine-integrated microfluidic chip IGRA read by a fluorescent microscope that has a 5 h sample-to-answer time and uses â¼25 µL of a fingerstick whole blood sample. Results from this assay correlated with those of a comparable clinical IGRA when used to evaluate the T-cell response to SARS-CoV-2 peptides in a population of vaccinated and/or infected individuals. Notably, this streamlined and inexpensive assay is suitable for high-throughput analyses in resource-limited settings for other infectious diseases.
ABSTRACT
Exosomes are membrane-defined extracellular vesicles (EVs) approximately 40-160 ânm in diameter that are found in all body fluids including blood, urine, and saliva. They act as important vehicles for intercellular communication between both local and distant cells and can serve as circulating biomarkers for disease diagnosis and prognosis. Exosomes play a key role in tumor metastasis, are abundant in biofluids, and stabilize biomarkers they carry, and thus can improve cancer detection, treatment monitoring, and cancer staging/prognosis. Despite their clinical potential, lack of sensitive/specific biomarkers and sensitive isolation/enrichment and analytical technologies has posed a barrier to clinical translation of exosomes. This review presents a critical overview of technologies now being used to detect tumor-derived exosome (TDE) biomarkers in clinical specimens that have potential for clinical translation.
ABSTRACT
Sensitive detection of extracellular vesicles (EVs) as emerging biomarkers has shown great promises for disease diagnosis. Plasmonic metal nanostructures conjugated with molecules that bind specific biomarker targets are widely used for EVs sensing but involve tradeoffs between particle-size-dependent signal intensity and conjugation efficiency. One solution to this problem would be to induce nucleation on nanoparticles that have successfully bound a target biomarker to permit in situ nanoparticle growth for signal amplification, but approaches that are evaluated to date require harsh conditions or lack nucleation specificity, prohibiting their effective use with most biological specimens. This study describes a one-step in situ strategy to induce monocrystalline copper shell growth on gold nanorod probes without decreasing signal by disrupting probe-target interactions or lipid bilayer integrity to enable EV biomarker detections. This approach increases the detected nanoparticle signal about two orders of magnitude after a 10 min copper nanoshell growth reaction. This has significant implications for improved disease detection, as indicated by the ability of a novel immunoassay using this approach to detect low abundance EVs carrying a pathogen-derived biomarker, after their direct capture from serum, to facilitate the diagnosis of tuberculosis cases in a diagnostically challenging pediatric cohort.
Subject(s)
Extracellular Vesicles , Nanoparticles , Humans , Child , Copper/metabolism , Biomarkers/analysis , Lipid Bilayers/metabolism , Extracellular Vesicles/metabolismABSTRACT
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common life-threatening lung diseases associated with acute and severe inflammation. Both have high mortality rates, and despite decades of research on clinical ALI/ARDS, there are no effective therapeutic strategies. Disruption of alveolar-capillary barrier integrity or activation of inflammatory responses leads to lung inflammation and injury. Recently, studies on the role of extracellular vesicles (EVs) in regulating normal and pathophysiologic cell activities, including inflammation and injury responses, have attracted attention. Injured and dysfunctional cells often secrete EVs into serum or bronchoalveolar lavage fluid with altered cargoes, which can be used to diagnose and predict the development of ALI/ARDS. EVs secreted by mesenchymal stem cells can also attenuate inflammatory reactions associated with cell dysfunction and injury to preserve or restore cell function, and thereby promote cell proliferation and tissue regeneration. This review focuses on the roles of EVs in the pathogenesis of pulmonary inflammation, particularly ALI/ARDS.
Subject(s)
Acute Lung Injury , Extracellular Vesicles , Mesenchymal Stem Cells , Pneumonia , Respiratory Distress Syndrome , Humans , Acute Lung Injury/etiology , Acute Lung Injury/therapy , Extracellular Vesicles/pathology , Mesenchymal Stem Cells/pathology , Mesenchymal Stem Cells/physiology , Respiratory Distress Syndrome/etiology , Respiratory Distress Syndrome/therapy , InflammationABSTRACT
Sensitive and specific blood-based assays for the detection of pulmonary and extrapulmonary tuberculosis would reduce mortality associated with missed diagnoses, particularly in children. Here we report a nanoparticle-enhanced immunoassay read by dark-field microscopy that detects two Mycobacterium tuberculosis virulence factors (the glycolipid lipoarabinomannan and its carrier protein) on the surface of circulating extracellular vesicles. In a cohort study of 147 hospitalized and severely immunosuppressed children living with HIV, the assay detected 58 of the 78 (74%) cases of paediatric tuberculosis, 48 of the 66 (73%) cases that were missed by microbiological assays, and 8 out of 10 (80%) cases undiagnosed during the study. It also distinguished tuberculosis from latent-tuberculosis infections in non-human primates. We adapted the assay to make it portable and operable by a smartphone. With further development, the assay may facilitate the detection of tuberculosis at the point of care, particularly in resource-limited settings.
Subject(s)
Extracellular Vesicles , Mycobacterium tuberculosis , Tuberculosis , Animals , Cohort Studies , Humans , Tuberculosis/diagnosis , Virulence FactorsABSTRACT
Identification of epitopes targeted following virus infection or vaccination can guide vaccine design and development of therapeutic interventions targeting functional sites, but can be laborious. Herein, we employed peptide microarrays to map linear peptide epitopes (LPEs) recognized following SARS-CoV-2 infection and vaccination. LPEs detected by nonhuman primate (NHP) and patient IgMs after SARS-CoV-2 infection extensively overlapped, localized to functionally important virus regions, and aligned with reported neutralizing antibody binding sites. Similar LPE overlap occurred after infection and vaccination, with LPE clusters specific to each stimulus, where strong and conserved LPEs mapping to sites known or likely to inhibit spike protein function. Vaccine-specific LPEs tended to map to sites known or likely to be affected by structural changes induced by the proline substitutions in the mRNA vaccine's S protein. Mapping LPEs to regions of known functional importance in this manner may accelerate vaccine evaluation and discovery of targets for site-specific therapeutic interventions.
ABSTRACT
BACKGROUND: Tuberculosis remains a leading cause of global mortality, especially for adults and children living with HIV (CLHIV) underdiagnosed by sputum-based assays. Non-sputum-based assays are needed to improve tuberculosis diagnosis and tuberculosis treatment monitoring. Our aim in this study was to determine whether ultrasensitive detection of Mycobacterium tuberculosis cell-free DNA (Mtb-cfDNA) in blood can diagnose tuberculosis and evaluate tuberculosis treatment responses. METHODS: In this molecular diagnostics study we analysed archived serum from two patient populations evaluated for tuberculosis in Eswatini and Kenya to detect Mtb-cfDNA, analysing serum from all individuals who had both sufficient serum volumes and clear diagnostic results. An optimised CRISPR-mediated tuberculosis (CRISPR-TB) assay was used to detect Mtb-cfDNA in serum at enrolment from adults and children with presumptive tuberculosis and their asymptomatic household contacts, and at enrolment and during tuberculosis treatment from a cohort of symptomatic CLHIV at high risk for tuberculosis, who provided longitudinal serum at enrolment and during tuberculosis treatment. FINDINGS: CRISPR-TB identified microbiologically and clinically confirmed tuberculosis cases in the predominantly HIV-negative Eswatini adult cohort with 96% sensitivity (27 [96%] of 28, 95% CI 80-100) and 94% specificity (16 [94%] of 17, 71-100), and with 83% sensitivity (5 [83%] of 6, 36-100) and 95% specificity (21 [95%] of 22, 77-100) in the paediatric cohort, including all six cases of extrapulmonary tuberculosis. In the Kenyan CLHIV cohort, CRISPR-TB detected all (13 [100%] of 13, 75-100) confirmed tuberculosis cases and 85% (39 [85%] of 46, 71-94) of unconfirmed tuberculosis cases diagnosed by non-microbiological clinical findings. CLHIV who were CRISPR-TB positive at enrolment had a 2·4-times higher risk of mortality by 6 months after enrolment. Mtb-cfDNA signal decreased after tuberculosis treatment initiation, with near or complete Mtb-cfDNA clearance by 6 months after tuberculosis treatment initiation. INTERPRETATION: CRISPR-mediated detection of circulating Mtb-cfDNA shows promise to increase the identification of paediatric tuberculosis and HIV-associated tuberculosis, and potential for early diagnosis and rapid monitoring of tuberculosis treatment responses. FUNDING: US Department of Defense, National Institute of Child Health and Human Development, National Institute of Allergy and Infectious Diseases, University of Washington Center for AIDS Research, and the Weatherhead Presidential Endowment fund.
Subject(s)
Cell-Free Nucleic Acids , HIV Infections , Mycobacterium tuberculosis , Tuberculosis, Lymph Node , Adult , Cell-Free Nucleic Acids/genetics , Child , Clustered Regularly Interspaced Short Palindromic Repeats , HIV Infections/diagnosis , Humans , Kenya/epidemiology , Mycobacterium tuberculosis/genetics , Pathology, Molecular , Sensitivity and Specificity , Tuberculosis, Lymph Node/genetics , United StatesABSTRACT
Rationale: Circulating pathogen-derived proteins can serve as useful biomarkers for infections but may be detected with poor sensitivity and specificity by standard immunoassays due to masking effects and cross-reactivity. Mass spectrometry (MS)-read immunoassays for biomarker-derived peptides can resolve these issues, but lack standard workflows to select species-specific peptides with strong MS signal that are suitable for antibody generation. Methods:Using a Mycobacterium tuberculosis (Mtb) protein as an example, candidate peptides were selected by length, species-specificity, MS intensity, and antigenicity score. MS data from spiked healthy serum was employed to define MS feature thresholds, including a novel measure of internal MS data correlation, to produce a peak detection algorithm. Results: This algorithm performed better in rejecting false positive signal than each of its criteria, including those currently employed for this purpose. Analysis of an Mtb peptide biomarker (CFP-10pep) by this approach identified tuberculosis cases not detected by microbiologic assays, including extrapulmonary tuberculosis and tuberculosis cases in children infected with HIV-1. Circulating CFP-10pep levels measured in a non-human primate model of tuberculosis distinguished disease from asymptomatic infection and tended to correspond with Mtb granuloma size, suggesting that it could also serve as a surrogate marker for Mtb burden and possibly treatment response. Conclusions: These biomarker selection and analysis approach appears to have strong potential utility for infectious disease diagnosis, including cryptic infections, and possibly to monitor changes in Mtb burden that may reflect disease progression or a response to treatment, which are critical needs for more effective disease control.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Animals , Biomarkers , Peptides , Sensitivity and Specificity , Tuberculosis/diagnosis , Tuberculosis/microbiologyABSTRACT
Mounting evidence indicates that SARS-CoV-2 can infect multiple systemic tissues, but few studies have evaluated SARS-CoV-2 RNA dynamics in multiple specimen types due to their reduced accessibility and diminished performance of RT-qPCR with non-respiratory specimens. Here, we employed an ultrasensitive CRISPR-RT-PCR assay to analyze longitudinal mucosal (nasal, buccal, pharyngeal, and rectal), plasma, and breath samples from SARS-CoV-2-infected non-human primates (NHPs) to detect dynamic changes in SARS-CoV-2 RNA level and distribution among these specimens. We observed that CRISPR-RT-PCR results consistently detected SARS-CoV-2 RNA in all sample types at most time points post-infection, and that SARS-CoV-2 infection dose and administration route did not markedly affect the CRISPR-RT-PCR signal detected in most specimen types. However, consistent RT-qPCR positive results were restricted to nasal, pharyngeal, and rectal swab samples, and tended to decrease earlier than CRISPR-RT-PCR results, reflecting lower assay sensitivity. SARS-CoV-2 RNA was detectable in both pulmonary and extrapulmonary specimens from early to late infection by CRISPR-RT-PCR, albeit with different abundance and kinetics, with SARS-CoV-2 RNA increases detected in plasma and rectal samples trailing those detected in upper respiratory tract samples. CRISPR-RT-PCR assays for SARS-CoV-2 RNA in non-respiratory specimens may thus permit direct diagnosis of suspected COVID-19 cases missed by RT-PCR, while tracking SARS-CoV-2 RNA in minimally invasive alternate specimens may better evaluate the progression and resolution of SARS-CoV-2 infections.
Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Humans , Primates , RNA, Viral/analysis , Sensitivity and Specificity , Serologic TestsABSTRACT
SARS-CoV-2 variants of concern (VOCs) that increase transmission or disease severity or reduce diagnostic or vaccine efficacy continue to emerge across the world. Current methods available to rapidly detect these can be resource intensive and thus sub-optimal for large-scale deployment needed during a pandemic response. Here, we describe a CRISPR-based assay that detects mutations in spike gene CRISPR PAM motif or seed regions to identify a pan-specific VOC single-nucleotide polymorphism (SNP)) ((D614G) and Alpha- and Delta-specific (S982A and D950N) SNPs. This assay exhibits good diagnostic sensitivity and strain specificity with nasal swabs and is designed for use in laboratory and point-of-care settings. This should enable rapid, high-throughput VOC identification required for surveillance and characterization efforts to inform clinical and public health decisions. Furthermore, the assay can be adapted to target similar SNPs associated with emerging SARS-CoV-2 VOCs, or other rapidly evolving viruses.