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1.
J Nanobiotechnology ; 20(1): 41, 2022 Jan 21.
Article in English | MEDLINE | ID: covidwho-1643157

ABSTRACT

Early detection of viral pathogens by DNA-sensors in clinical samples, contaminated foods, soil or water can dramatically improve clinical outcomes and reduce the socioeconomic impact of diseases such as COVID-19. Clustered regularly interspaced short palindromic repeat (CRISPR) and its associated protein Cas12a (previously known as CRISPR-Cpf1) technology is an innovative new-generation genomic engineering tool, also known as 'genetic scissors', that has demonstrated the accuracy and has recently been effectively applied as appropriate (E-CRISPR) DNA-sensor to detect the nucleic acid of interest. The CRISPR-Cas12a from Prevotella and Francisella 1 are guided by a short CRISPR RNA (gRNA). The unique simultaneous cis- and trans- DNA cleavage after target sequence recognition at the PAM site, sticky-end (5-7 bp) employment, and ssDNA/dsDNA hybrid cleavage strategies to manipulate the attractive nature of CRISPR-Cas12a are reviewed. DNA-sensors based on the CRISPR-Cas12a technology for rapid, robust, sensitive, inexpensive, and selective detection of virus DNA without additional sample purification, amplification, fluorescent-agent- and/or quencher-labeling are relevant and becoming increasingly important in industrial and medical applications. In addition, CRISPR-Cas12a system shows great potential in the field of E-CRISPR-based bioassay research technologies. Therefore, we are highlighting insights in this research direction.


Subject(s)
CRISPR-Cas Systems/physiology , DNA, Viral/isolation & purification , Nucleic Acid Amplification Techniques , Animals , Biosensing Techniques/methods , Biosensing Techniques/trends , COVID-19/virology , DNA, Viral/analysis , Environmental Pollutants/analysis , Environmental Pollutants/isolation & purification , Food Contamination/analysis , Humans , Molecular Typing/methods , Molecular Typing/trends , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/trends , SARS-CoV-2/genetics , Virology/methods , Virology/trends , Virus Diseases/classification , Virus Diseases/diagnosis , Virus Diseases/virology
2.
J Am Chem Soc ; 143(41): 17004-17014, 2021 10 20.
Article in English | MEDLINE | ID: covidwho-1461966

ABSTRACT

Rapid screening of infected individuals from a large population is an effective means in epidemiology, especially to contain outbreaks such as COVID-19. The gold standard assays for COVID-19 diagnostics are mainly based on the reverse transcription polymerase chain reaction, which mismatches the requirements for wide-population screening due to time-consuming nucleic acid extraction and amplification procedures. Here, we report a direct nucleic acid assay by using a graphene field-effect transistor (g-FET) with Y-shaped DNA dual probes (Y-dual probes). The assay relies on Y-dual probes modified on g-FET simultaneously targeting ORF1ab and N genes of SARS-CoV-2 nucleic acid, enabling high a recognition ratio and a limit of detection (0.03 copy µL-1) 1-2 orders of magnitude lower than existing nucleic acid assays. The assay realizes the fastest nucleic acid testing (∼1 min) and achieves direct 5-in-1 pooled testing for the first time. Owing to its rapid, ultrasensitive, easily operated features as well as capability in pooled testing, it holds great promise as a comprehensive tool for population-wide screening of COVID-19 and other epidemics.


Subject(s)
DNA Probes , DNA, Viral/analysis , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , COVID-19/diagnosis , COVID-19/virology , Graphite/chemistry , Humans , Limit of Detection
3.
Cancer Prev Res (Phila) ; 14(10): 919-926, 2021 10.
Article in English | MEDLINE | ID: covidwho-1450634

ABSTRACT

The World Health Organization global call to eliminate cervical cancer encourages countries to consider introducing or improving cervical cancer screening programs. Brazil's Unified Health System (SUS) is among the world's largest public health systems offering free cytology testing, follow-up colposcopy, and treatment. Yet, health care networks across the country have unequal infrastructure, human resources, equipment, and supplies resulting in uneven program performance and large disparities in cervical cancer incidence and mortality. An effective screening program needs multiple strategies feasible for each community's reality, facilitating coverage and follow-up adherence. Prioritizing those at highest risk with tests that better stratify risk will limit inefficiencies, improving program impact across different resource settings. Highly sensitive human papillomavirus (HPV)-DNA testing performs better than cytology and, with self-collection closer to homes and workplaces, improves access, even in remote regions. Molecular triage strategies like HPV genotyping can identify from the same self-collected sample, those at highest risk requiring follow-up. If proven acceptable, affordable, cost-effective, and efficient in the Brazilian context, these strategies would increase coverage while removing the need for speculum exams for routine screening and reducing follow-up visits. SUS could implement a nationwide organized program that accommodates heterogenous settings across Brazil, informing a variety of screening programs worldwide.


Subject(s)
COVID-19/complications , Cytodiagnosis/methods , Early Detection of Cancer/methods , Papillomaviridae/isolation & purification , Papillomavirus Infections/complications , SARS-CoV-2/isolation & purification , Uterine Cervical Neoplasms/diagnosis , Brazil/epidemiology , DNA, Viral/analysis , DNA, Viral/genetics , Female , Humans , Papillomavirus Infections/genetics , Papillomavirus Infections/virology , Uterine Cervical Neoplasms/epidemiology , Uterine Cervical Neoplasms/virology
6.
J Mater Chem B ; 9(28): 5636-5644, 2021 07 21.
Article in English | MEDLINE | ID: covidwho-1291040

ABSTRACT

DNA polymerization is of high specificity in vivo. However, its specificity is much lower in vitro, which limits advanced applications of DNA polymerization in ultrasensitive nucleic acid detection. Herein, we report a unique mechanism of single selenium-atom modified dNTP (dNTPαSe) to enhance polymerization specificity. We have found that both dNTPαSe (approximately 660 fold) and Se-DNA (approximately 2.8 fold) have lower binding affinity to DNA polymerase than canonical ones, and the Se-DNA duplex has much lower melting-temperature (Tm) than the corresponding canonical DNA duplex. The reduced affinity and Tm can destabilize the substrate-primer-template-enzyme assembly, thereby largely slowing down the mismatch of DNA polymerization and enhancing the amplification specificity and in turn detection sensitivity. Furthermore, the Se-strategy enables us to develop the selenium enhanced specific isothermal amplification (SEA) for nucleic acid detection with high specificity and sensitivity (up to detection of single-digit copies), allowing convenient detection of clinical HPV and COVID-19 viruses in the low-copy number. Clearly, we have discovered the exciting mechanism for enhancing DNA polymerization accuracy, amplification specificity and detection sensitivity by SEA, up to two orders of magnitude higher.


Subject(s)
DNA, Viral/analysis , Human papillomavirus 16/isolation & purification , Phosphates/chemistry , SARS-CoV-2/isolation & purification , Selenium/chemistry , DNA, Viral/biosynthesis , Human papillomavirus 16/metabolism , Humans , Nucleic Acid Amplification Techniques , Polymerization , SARS-CoV-2/metabolism , Temperature
7.
Biochem Biophys Res Commun ; 567: 195-200, 2021 08 27.
Article in English | MEDLINE | ID: covidwho-1263226

ABSTRACT

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets. After evaluating the concentration of each component, the uvsY, gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the balance between the binding and dissociation of uvsX and DNA primer was precisely adjusted. Under the optimized condition, 60 copies of the target DNA were specifically detected. Detection of 60 copies of RNA was also achieved. Our results prove the fabrication flexibility of RPA reagents, leading to an expansion of the use of RPA in various fields.


Subject(s)
DNA, Viral/analysis , DNA-Directed DNA Polymerase/metabolism , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , RNA, Viral/analysis , Recombinases/metabolism , SARS-CoV-2/genetics , Statistics as Topic , DNA Primers/metabolism , DNA-Binding Proteins/metabolism , Membrane Proteins/metabolism , SARS-CoV-2/isolation & purification , Viral Proteins/metabolism
8.
Transplantation ; 105(9): 2072-2079, 2021 09 01.
Article in English | MEDLINE | ID: covidwho-1254952

ABSTRACT

BACKGROUND: The impacts of COVID-19 on lung allograft function, rejection, secondary infection, and clinical outcomes in lung transplant recipients (LTRs) remain unknown. METHODS: A 1:2 matched case-control study was performed to evaluate rehospitalization, lung allograft function, and secondary infections up to 90 d after COVID-19 diagnosis (or index dates for controls). RESULTS: Twenty-four LTRs with COVID-19 (cases) and 48 controls were identified. Cases and controls had similar baseline characteristics and lung allograft function. LTRs with COVID-19 had higher incidence of secondary bacterial infection (29.2% versus 6.3%, P = 0.008), readmission (29.2% versus 10.4%, P = 0.04), and for-cause bronchoscopy (33.3% versus 12.5%, P = 0.04) compared with controls. At d 90, mortality in cases versus controls was 8.3% versus 2.1% (P = 0.21), incidence of invasive fungal infections in cases versus controls was 20.8% versus 8.3% (P = 0.13) and forced expiratory volume in 1 s (FEV1) decline ≥10% from baseline occurred in 19% of cases versus 12.2% of controls (P = 0.46). No acute cellular rejection, acute antibody-mediated rejection, or new donor-specific anti-HLA antibodies were observed among cases or controls within 90 d post index date. CONCLUSIONS: We found LTRs with COVID-19 were at risk to develop secondary infections and rehospitalization post COVID-19, compared with controls. While we did not observe post viral acute cellular rejection or antibody-mediated rejection, further studies are needed to understand if LTRs with COVID-19 who did not recover baseline lung function within 90 d have developed chronic lung allograft dysfunction stage progression.


Subject(s)
COVID-19/epidemiology , Graft Rejection/epidemiology , Lung Diseases/surgery , Transplant Recipients , Adult , Aged , Allografts , Comorbidity , DNA, Viral/analysis , Female , Follow-Up Studies , Humans , Incidence , Lung Diseases/epidemiology , Lung Transplantation , Male , Middle Aged , Pandemics , Retrospective Studies , Risk Factors , SARS-CoV-2/genetics , United States/epidemiology
9.
Am J Respir Crit Care Med ; 203(9): 1070-1087, 2021 05 01.
Article in English | MEDLINE | ID: covidwho-1223640

ABSTRACT

Background: This document provides evidence-based clinical practice guidelines on the diagnostic utility of nucleic acid-based testing of respiratory samples for viral pathogens other than influenza in adults with suspected community-acquired pneumonia (CAP).Methods: A multidisciplinary panel developed a Population-Intervention-Comparison-Outcome question, conducted a pragmatic systematic review, and applied Grading of Recommendations, Assessment, Development, and Evaluation methodology for clinical recommendations.Results: The panel evaluated the literature to develop recommendations regarding whether routine diagnostics should include nucleic acid-based testing of respiratory samples for viral pathogens other than influenza in suspected CAP. The evidence addressing this topic was generally adjudicated to be of very low quality because of risk of bias and imprecision. Furthermore, there was little direct evidence supporting a role for routine nucleic acid-based testing of respiratory samples in improving critical outcomes such as overall survival or antibiotic use patterns. However, on the basis of direct and indirect evidence, recommendations were made for both outpatient and hospitalized patients with suspected CAP. Testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was not addressed in the literature at the time of the evidence review.Conclusions: The panel formulated and provided their rationale for recommendations on nucleic acid-based diagnostics for viral pathogens other than influenza for patients with suspected CAP.


Subject(s)
Community-Acquired Infections/virology , DNA, Viral/analysis , Pneumonia/virology , Societies, Medical , Viruses/genetics , Community-Acquired Infections/diagnosis , Humans , Pneumonia/diagnosis
10.
Viruses ; 13(5)2021 04 29.
Article in English | MEDLINE | ID: covidwho-1217117

ABSTRACT

The availability of simple SARS-CoV-2 detection methods is crucial to contain the COVID-19 pandemic. This study examined whether a commercial LAMP assay can reliably detect SARS-CoV-2 genomes directly in respiratory samples without having to extract nucleic acids (NA) beforehand. Nasopharyngeal swabs (NPS, n = 220) were tested by real-time reverse transcription (RT)-PCR and with the LAMP assay. For RT-PCR, NA were investigated. For LAMP, NA from 26 NPS in viral transport medium (VTM) were tested. The other 194 NPS were analyzed directly without prior NA extraction (140 samples in VTM; 54 dry swab samples stirred in phosphate buffered saline). Ten NPS were tested directly by LAMP using a sous-vide cooking unit. The isothermal assay demonstrated excellent specificity (100%) but moderate sensitivity (68.8%), with a positive predictive value of 1 and a negative predictive value of 0.65 for direct testing of NPS in VTM. The use of dry swabs, even without NA extraction, improved the analytical sensitivity; up to 6% of samples showed signs of inhibition. LAMP could be performed successfully with a sous-vide cooking unit. This technique is very fast, requires little laboratory resources, and can replace rapid antigen tests or verify reactive rapid tests on-site.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , DNA, Viral/analysis , Molecular Diagnostic Techniques/methods , Nasopharynx/virology , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/isolation & purification , Humans , Sensitivity and Specificity , Specimen Handling
12.
J Pediatr ; 232: 287-289.e4, 2021 05.
Article in English | MEDLINE | ID: covidwho-1126937

ABSTRACT

We conducted a multicenter clinical validity study of the Panbio coronavirus disease 2019 Antigen Rapid Test of nasopharyngeal samples in pediatric patients with coronavirus disease 2019-compatible symptoms of ≤5 days of evolution. Our study showed limited accuracy in nasopharyngeal antigen testing: overall sensitivity was 45.4%, and 99.8% of specificity, positive-predictive value was 92.5%.


Subject(s)
Antigens, Viral/analysis , COVID-19/diagnosis , DNA, Viral/analysis , Nasopharynx/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Adolescent , COVID-19/virology , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Pandemics , Reproducibility of Results , SARS-CoV-2/immunology
13.
Anal Chem ; 93(9): 4154-4159, 2021 03 09.
Article in English | MEDLINE | ID: covidwho-1108879

ABSTRACT

Chip-scale SARS-CoV-2 testing was demonstrated using silicon nitride (Si3N4) nanoslot fluidic waveguides to detect a tagged oligonucleotide with a coronavirus DNA sequence. The slot waveguides were fabricated using complementary metal-oxide-semiconductor (CMOS) fabrication processes, including multiscale lithography and selective reactive ion etching (RIE), forming femtoliter fluidic channels. Finite difference method (FDM) simulation was used to calculate the optical field distribution of the waveguide mode when the waveguide sensor was excited by transverse electric (TE) and transverse magnetic (TM) polarized light. For the TE polarization, a strong optical field was created in the slot region and its field intensity was 14× stronger than the evanescent sensing field from the TM polarization. The nanoscale confinement of the optical sensing field significantly enhanced the light-analyte interaction and improved the optical sensitivity. The sensitivity enhancement was experimentally demonstrated by measuring the polarization-dependent fluorescence emission from the tagged oligonucleotide. The photonic chips consisting of femtoliter Si3N4 waveguides provide a low-cost and high throughput platform for real-time virus identification, which is critical for point-of-care (PoC) diagnostic applications.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/virology , DNA, Viral/analysis , Nanoparticles/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Silicon Compounds/chemistry , Humans , Optics and Photonics , Point-of-Care Systems , Refractometry , Semiconductors , Sensitivity and Specificity
14.
Talanta ; 227: 122154, 2021 May 15.
Article in English | MEDLINE | ID: covidwho-1078200

ABSTRACT

Infectious diseases caused by viruses such as SARS-CoV-2 and HPV have greatly endangered human health. The nucleic acid detection is essential for the early diagnosis of diseases. Here, we propose a method called PLCR (PfAgo coupled with modified Ligase Chain Reaction for nucleic acid detection) which utilizes PfAgo to only use DNA guides longer than 14-mer to specifically cleave DNA and LCR to precisely distinguish single-base mismatch. PLCR can detect DNA or RNA without PCR at attomolar sensitivities, distinguish single base mutation between the genome of wild type SARS-CoV-2 and its mutant spike D614G, effectively distinguish the novel coronavirus from other coronaviruses and finally achieve multiplexed detection in 70 min. Additionally, LCR products can be directly used as DNA guides without additional input guides to simplify primer design. With desirable sensitivity, specificity and simplicity, the method can be extended for detecting other pathogenic microorganisms.


Subject(s)
Argonaute Proteins/chemistry , DNA, Viral/analysis , Ligase Chain Reaction/methods , Pyrococcus furiosus/enzymology , RNA, Viral/analysis , Alphapapillomavirus/chemistry , Alphapapillomavirus/isolation & purification , COVID-19/diagnosis , DNA, Viral/chemistry , Humans , Limit of Detection , Mutation , Papillomavirus Infections/diagnosis , RNA, Viral/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics
15.
PLoS One ; 15(10): e0241542, 2020.
Article in English | MEDLINE | ID: covidwho-1067389

ABSTRACT

Oral swabs are emerging as a non-invasive sample type for diagnosing infectious diseases including Ebola, tuberculosis (TB), and COVID-19. To assure proper sample collection, sample adequacy controls (SACs) are needed that detect substances indicative of samples collected within the oral cavity. This study evaluated two candidate SACs for this purpose. One detected representative oral microbiota (Streptococcus species DNA) and the other, human cells (human mitochondrial DNA, mtDNA). Quantitative PCR (qPCR) assays for the two target cell types were applied to buccal swabs (representing samples collected within the oral cavity) and hand swabs (representing improperly collected samples) obtained from 51 healthy U.S. volunteers. Quantification cycle (Cq) cutoffs that maximized Youden's index were established for each assay. The streptococcal target at a Cq cutoff of ≤34.9 had 99.0% sensitivity and specificity for oral swab samples, whereas human mtDNA perfectly distinguished between hand and mouth swabs with a Cq cutoff of 31.3. The human mtDNA test was then applied to buccal, tongue, and gum swabs that had previously been collected from TB patients and controls in South Africa, along with "air swabs" collected as negative controls (total N = 292 swabs from 71 subjects). Of these swabs, 287/292 (98%) exhibited the expected Cq values. In a paired analysis the three oral sites yielded indistinguishable amounts of human mtDNA, however PurFlockTM swabs collected slightly more human mtDNA than did OmniSwabsTM (p = 0.012). The results indicate that quantification of human mtDNA cannot distinguish swabs collected from different sites within the mouth. However, it can reliably distinguish oral swabs from swabs that were not used orally, which makes it a useful SAC for oral swab-based diagnosis.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Specimen Handling/methods , Adult , COVID-19/epidemiology , COVID-19/transmission , COVID-19/virology , DNA, Mitochondrial/analysis , DNA, Mitochondrial/genetics , DNA, Viral/analysis , DNA, Viral/genetics , Diagnostic Tests, Routine/methods , Female , Humans , Male , Mouth/virology , Real-Time Polymerase Chain Reaction , Reference Standards , Sensitivity and Specificity , South Africa/epidemiology , Washington/epidemiology
16.
J Chem Inf Model ; 60(12): 6427-6437, 2020 12 28.
Article in English | MEDLINE | ID: covidwho-1065779

ABSTRACT

CRISPR-Cas12a is a genome-editing system, recently also harnessed for nucleic acid detection, which is promising for the diagnosis of the SARS-CoV-2 coronavirus through the DETECTR technology. Here, a collective ensemble of multimicrosecond molecular dynamics characterizes the key dynamic determinants allowing nucleic acid processing in CRISPR-Cas12a. We show that DNA binding induces a switch in the conformational dynamics of Cas12a, which results in the activation of the peripheral REC2 and Nuc domains to enable cleavage of nucleic acids. The simulations reveal that large-amplitude motions of the Nuc domain could favor the conformational activation of the system toward DNA cleavages. In this process, the REC lobe plays a critical role. Accordingly, the joint dynamics of REC and Nuc shows the tendency to prime the conformational transition of the DNA target strand toward the catalytic site. Most notably, the highly coupled dynamics of the REC2 region and Nuc domain suggests that REC2 could act as a regulator of the Nuc function, similar to what was observed previously for the HNH domain in the CRISPR-associated nuclease Cas9. These mutual domain dynamics could be critical for the nonspecific binding of DNA and thereby for the underlying mechanistic functioning of the DETECTR technology. Considering that REC is a key determinant in the system's specificity, our findings provide a rational basis for future biophysical studies aimed at characterizing its function in CRISPR-Cas12a. Overall, our outcomes advance our mechanistic understanding of CRISPR-Cas12a and provide grounds for novel engineering efforts to improve genome editing and viral detection.


Subject(s)
COVID-19/diagnosis , DNA, Viral/analysis , DNA, Viral/genetics , SARS-CoV-2/genetics , CRISPR-Cas Systems , Catalytic Domain , DNA Cleavage , Gene Editing , Humans , Molecular Dynamics Simulation , Nucleic Acid Conformation , Phase Transition , Substrate Specificity
17.
Am J Respir Crit Care Med ; 203(9): 1112-1118, 2021 05 01.
Article in English | MEDLINE | ID: covidwho-1060897

ABSTRACT

Rationale: Patients with severe coronavirus disease (COVID-19) require supplemental oxygen and ventilatory support. It is unclear whether some respiratory support devices may increase the dispersion of infectious bioaerosols and thereby place healthcare workers at increased risk of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).Objectives: To quantitatively compare viral dispersion from invasive and noninvasive respiratory support modalities.Methods: This study used a simulated ICU room with a breathing-patient simulator exhaling nebulized bacteriophages from the lower respiratory tract with various respiratory support modalities: invasive ventilation (through an endotracheal tube with an inflated cuff connected to a mechanical ventilator), helmet ventilation with a positive end-expiratory pressure (PEEP) valve, noninvasive bilevel positive-pressure ventilation, nonrebreather face masks, high-flow nasal oxygen (HFNO), and nasal prongs.Measurements and Main Results: Invasive ventilation and helmet ventilation with a PEEP valve were associated with the lowest bacteriophage concentrations in the air, and HFNO and nasal prongs were associated with the highest concentrations. At the intubating position, bacteriophage concentrations associated with HFNO (2.66 × 104 plaque-forming units [PFU]/L of air sampled), nasal prongs (1.60 × 104 PFU/L of air sampled), nonrebreather face masks (7.87 × 102 PFU/L of air sampled), and bilevel positive airway pressure (1.91 × 102 PFU/L of air sampled) were significantly higher than those associated with invasive ventilation (P < 0.05 for each). The difference between bacteriophage concentrations associated with helmet ventilation with a PEEP valve (4.29 × 10-1 PFU/L of air sampled) and bacteriophage concentrations associated with invasive ventilation was not statistically significant.Conclusions: These findings highlight the potential differential risk of dispersing virus among respiratory support devices and the importance of appropriate infection prevention and control practices and personal protective equipment for healthcare workers when caring for patients with transmissible respiratory viral infections such as SARS-CoV-2.


Subject(s)
Critical Care/methods , DNA, Viral/analysis , Disease Transmission, Infectious/prevention & control , Respiratory Insufficiency/therapy , Ventilators, Mechanical/adverse effects , Virus Diseases/virology , Viruses/genetics , Humans , Virus Diseases/prevention & control , Virus Diseases/transmission
18.
Clin Chem Lab Med ; 59(5): 987-994, 2021 04 27.
Article in English | MEDLINE | ID: covidwho-1024424

ABSTRACT

OBJECTIVES: The qualitative results of SARS-CoV-2 specific real-time reverse transcription (RT) PCR are used for initial diagnosis and follow-up of Covid-19 patients and asymptomatic virus carriers. However, clinical decision-making and health management policies often are based additionally on cycle threshold (Ct) values (i.e., quantitative results) to guide patient care, segregation and discharge management of individuals testing positive. Therefore, an analysis of inter-protocol variability is needed to assess the comparability of the quantitative results. METHODS: Ct values reported in a SARS-CoV-2 virus genome detection external quality assessment challenge were analyzed. Three positive and two negative samples were distributed to participating test laboratories. Qualitative results (positive/negative) and quantitative results (Ct values) were assessed. RESULTS: A total of 66 laboratories participated, contributing results from 101 distinct test systems and reporting Ct values for a total of 92 different protocols. In all three positive samples, the means of the Ct values for the E-, N-, S-, RdRp-, and ORF1ab-genes varied by less than two cycles. However, 7.7% of reported results deviated by more than ±4.0 (maximum 18.0) cycles from the respective individual means. These larger deviations appear to be systematic errors. CONCLUSIONS: In an attempt to use PCR diagnostics beyond the identification of infected individuals, laboratories are frequently requested to report Ct values along with a qualitative result. This study highlights the limitations of interpreting Ct values from the various SARS-CoV genome detection protocols and suggests that standardization is necessary in the reporting of Ct values with respect to the target gene.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , DNA, Viral/analysis , Genome, Viral , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/chemistry , COVID-19/diagnosis , COVID-19 Nucleic Acid Testing/statistics & numerical data , False Negative Reactions , False Positive Reactions , Humans , Reverse Transcriptase Polymerase Chain Reaction/statistics & numerical data
19.
Chem Commun (Camb) ; 57(9): 1125-1128, 2021 Feb 02.
Article in English | MEDLINE | ID: covidwho-1012695

ABSTRACT

Two fragments of the COVID-19 genome (specific and homologous) were used as two inputs to construct an AND logic gate for COVID-19 detection based on exonuclease III and DNAzyme. The detection sensitivity of the assay can reach fM levels. Satisfactory recovery values were obtained in real sample analysis.


Subject(s)
COVID-19 Testing , COVID-19/diagnosis , DNA, Viral/analysis , SARS-CoV-2/genetics , Saliva/virology , DNA, Catalytic , DNA, Viral/blood , DNA, Viral/urine , Exodeoxyribonucleases , Genome, Viral , Humans , Logic
20.
Int J Infect Dis ; 104: 315-319, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-988038

ABSTRACT

OBJECTIVES: The immunologic profile and opportunistic viral DNA increase were monitored in Italian patients with COVID-19 in order to identify markers of disease severity. METHODS: A total of 104 patients infected with SARS-CoV-2 were evaluated in the study. Of them, 42/104 (40.4%) were hospitalized in an intensive care unit (ICU) and 62/104(59.6%) in a sub-intensive care unit (SICU). Human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV), Parvovirus B19 and Human Herpesvirus 6 virus reactivations were determined by real-time PCR, and lymphocyte subpopulation counts were determined by flow cytometry. RESULTS: Among opportunistic viruses, only EBV was consistently detected. EBV DNA was observed in 40/42 (95.2%) of the ICU patients and in 51/61 (83.6%) of the SICU patients. Comparing the two groups of patients, the EBV DNA median level among ICU patients was significantly higher than that observed in SICU patients. In parallel, a significant reduction of CD8 T cell and NK count in ICU patients as compared with SICU patients was observed (p<0.05). In contrast, B cell count was significantly increased in ICU patients (p=0.0172). CONCLUSIONS: A correlation between reduced CD8+ T cells and NK counts, EBV DNA levels and COVID-19 severity was observed. Other opportunistic viral infections were not observed. The relationship between EBV load and COVID-19 severity should be further evaluated in longitudinal studies.


Subject(s)
COVID-19/complications , Epstein-Barr Virus Infections/virology , Herpesvirus 4, Human/isolation & purification , SARS-CoV-2 , Viral Load , Aged , Aged, 80 and over , CD8-Positive T-Lymphocytes/virology , COVID-19/virology , DNA, Viral/analysis , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/diagnosis , Female , Herpesvirus 4, Human/genetics , Humans , Intensive Care Units , Killer Cells, Natural/virology , Lymphocyte Count , Lymphocyte Subsets/virology , Male , Middle Aged , Opportunistic Infections , Real-Time Polymerase Chain Reaction
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