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1.
Medicine (Baltimore) ; 100(21): e26143, 2021 May 28.
Article in English | MEDLINE | ID: covidwho-2191018

ABSTRACT

INTRODUCTION: Coronavirus disease 2019 (COVID-19) is a rapidly emerging infectious respiratory disease caused by severe acute respiratory syndrome coronavirus 2. Currently, more than 100 million cases of COVID-19 have been confirmed worldwide, with over 2.4 million mortalities. The pandemic affects people of all ages but older individuals and those with severe chronic illnesses, including cancer patients, are at higher risk. PATIENT CONCERNS: The impact of cancer treatment on the progression of COVID-19 is unclear. Therefore, we assessed the effects of chemotherapy on COVID-19 outcomes for 2 cancer patients. On January 24, 2020, a level I response to a major public health emergency was initiated in Hubei Province, China, which includes Enshi Autonomous Prefecture that has a population of 4.026 million people. As of April 30, 2020, 252 confirmed cases of COVID-19 and 11 asymptomatic carriers were identified in Enshi. DIAGNOSIS: Among the confirmed cases and asymptomatic carriers, 2 patients were identified who were previously diagnosed with malignant tumors, including one with hepatocellular carcinoma and the other with cardia carcinoma. INTERVENTIONS: These 2 patients were receiving or just completed chemotherapy at the time of their COVID-19 diagnosis. OUTCOMES: Both patients were followed and presented favorable outcomes. The positive outcomes for these 2 patients could be partially explained by their recent chemotherapy that impacted their immune status. Also, their relatively younger ages and lack of comorbidities were likely factors in their successful recovery from COVID-19. CONCLUSIONS: Anticancer treatment might enhance a patient's ability to respond favorably to COVID-19 infection. However, anticancer treatment is likely to impact immune function differently in different individuals, which can influence disease outcomes.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , COVID-19/immunology , Liver Neoplasms/drug therapy , SARS-CoV-2/immunology , Stomach Neoplasms/drug therapy , Adult , COVID-19/complications , COVID-19/diagnosis , COVID-19/drug therapy , COVID-19 Nucleic Acid Testing , Cyclobutanes/therapeutic use , Docetaxel/therapeutic use , Drug Therapy, Combination/methods , Humans , Liver Neoplasms/complications , Liver Neoplasms/immunology , Lung/diagnostic imaging , Male , Middle Aged , Organoplatinum Compounds/therapeutic use , RNA, Viral/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sorafenib/therapeutic use , Stomach Neoplasms/complications , Stomach Neoplasms/immunology , Tomography, X-Ray Computed , Treatment Outcome
2.
Medicine (Baltimore) ; 100(21): e26034, 2021 May 28.
Article in English | MEDLINE | ID: covidwho-2191014

ABSTRACT

ABSTRACT: To determine the role of ultra-low dose chest computed tomography (uld CT) compared to chest radiographs in patients with laboratory-confirmed early stage SARS-CoV-2 pneumonia.Chest radiographs and uld CT of 12 consecutive suspected SARS-CoV-2 patients performed up to 48 hours from hospital admission were reviewed by 2 radiologists. Dosimetry and descriptive statistics of both modalities were analyzed.On uld CT, parenchymal abnormalities compatible with SARS-CoV-2 pneumonia were detected in 10/12 (83%) patients whereas on chest X-ray in, respectively, 8/12 (66%) and 5/12 (41%) patients for reader 1 and 2. The average increment of diagnostic performance of uld CT compared to chest X-ray was 29%. The average effective dose was, respectively, of 0.219 and 0.073 mSv.Uld CT detects substantially more lung injuries in symptomatic patients with suspected early stage SARS-CoV-2 pneumonia compared to chest radiographs, with a significantly better inter-reader agreement, at the cost of a slightly higher equivalent radiation dose.


Subject(s)
COVID-19/diagnosis , Lung/diagnostic imaging , Radiography, Thoracic/statistics & numerical data , SARS-CoV-2/isolation & purification , Tomography, X-Ray Computed/statistics & numerical data , Adult , Aged , Aged, 80 and over , COVID-19/virology , COVID-19 Nucleic Acid Testing , Female , Humans , Male , Middle Aged , Observer Variation , Predictive Value of Tests , RNA, Viral/isolation & purification , Radiation Dosage , Radiography, Thoracic/adverse effects , Radiography, Thoracic/methods , Radiometry/statistics & numerical data , Retrospective Studies , SARS-CoV-2/genetics , Tomography, X-Ray Computed/adverse effects , Tomography, X-Ray Computed/methods
3.
Medicine (Baltimore) ; 100(21): e25645, 2021 May 28.
Article in English | MEDLINE | ID: covidwho-2190994

ABSTRACT

ABSTRACT: Since December 2019, pneumonia caused by a novel coronavirus (SARS-CoV-2), namely 2019 novel coronavirus disease (COVID-19), has rapidly spread from Wuhan city to other cities across China. The present study was designed to describe the epidemiology, clinical characteristics, treatment, and prognosis of 74 hospitalized patients with COVID-19.Clinical data of 74 COVID-19 patients were collected to analyze the epidemiological, demographic, laboratory, radiological, and treatment data. Thirty-two patients were followed up and tested for the presence of the viral nucleic acid and by pulmonary computed tomography (CT) scan at 7 and 14 days after they were discharged.Among all COVID-19 patients, the median incubation period for patients and the median period from symptom onset to admission was all 6 days; the median length of hospitalization was 13 days. Fever symptoms were presented in 83.78% of the patients, and the second most common symptom was cough (74.32%), followed by fatigue and expectoration (27.03%). Inflammatory indicators, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) of the intensive care unit (ICU) patients were significantly higher than that of the non-ICU patients (P < .05). However, 50.00% of the ICU patients had their the ratio of T helper cells to cytotoxic T cells (CD4/CD8) ratio lower than 1.1, whose proportion is much higher than that in non-ICU patients (P < .01).Compared with patients in Wuhan, COVID-19 patients in Anhui Province seemed to have milder symptoms of infection, suggesting that there may be some regional differences in the transmission of SARS-CoV-2 between different cities.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/diagnosis , Cough/epidemiology , Fever/epidemiology , Hyperbaric Oxygenation/statistics & numerical data , Adolescent , Adult , Aged , Antibiotic Prophylaxis/statistics & numerical data , Blood Sedimentation , C-Reactive Protein/analysis , COVID-19/complications , COVID-19/epidemiology , COVID-19/therapy , COVID-19 Nucleic Acid Testing , Child , Child, Preschool , China/epidemiology , Cities/epidemiology , Cough/blood , Cough/therapy , Cough/virology , Female , Fever/blood , Fever/therapy , Fever/virology , Follow-Up Studies , Geography , Humans , Length of Stay/statistics & numerical data , Lung/diagnostic imaging , Male , Middle Aged , RNA, Viral/isolation & purification , Retrospective Studies , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Severity of Illness Index , Tomography, X-Ray Computed , Young Adult
7.
BMC Infect Dis ; 22(1): 697, 2022 Aug 18.
Article in English | MEDLINE | ID: covidwho-1993335

ABSTRACT

BACKGROUND: High cost of commercial RNA extraction kits limits the testing efficiency of SARS-CoV-2. Here, we developed a simple nucleic acid extraction method for the detection of SARS-CoV-2 directly from nasopharyngeal swab samples. METHODS: A pH sensitive dye was used as the end point detection method. The obvious colour changes between positive and negative reactions eliminates the need of other equipment. RESULTS: Clinical testing using 260 samples showed 92.7% sensitivity (95% CI 87.3-96.3%) and 93.6% specificity (95% CI 87.3-97.4%) of RT-LAMP. CONCLUSIONS: The simple RNA extraction method minimizes the need for any extensive laboratory set-up. We suggest combining this simple nucleic acid extraction method and RT-LAMP technology as the point-of care diagnostic tool.


Subject(s)
COVID-19 Testing , COVID-19 , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , SARS-CoV-2 , COVID-19/diagnosis , COVID-19/virology , COVID-19 Testing/methods , Humans , Molecular Diagnostic Techniques/methods , Nasopharynx/virology , Nucleic Acid Amplification Techniques/methods , Point-of-Care Systems , RNA, Viral/analysis , RNA, Viral/genetics , RNA, Viral/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
8.
Clin Chim Acta ; 532: 181-187, 2022 Jul 01.
Article in English | MEDLINE | ID: covidwho-1966413

ABSTRACT

BACKGROUND: SARS-CoV-2 is an RNA virus that primarily causes respiratory disease; however, infection of other tissue has been reported. Evaluation of SARS-CoV-2 in tissue specimens may increase understanding of SARS-CoV-2 pathobiology. MATERIALS AND METHODS: A qualitative test for detection of SARS-CoV-2 in formalin-fixed paraffin-embedded (FFPE) tissues was developed and validated using droplet digital PCR (ddPCR), which has a lower limit of detection than reverse transcription (RT)-qPCR. After extraction of total RNA from unstained FFPE tissue, SARS-CoV-2 nucleocapsid (N1, N2) target sequences were amplified and quantified, along with human RPP30 as a control using the Bio-Rad SARS-CoV-2 ddPCR kit. RESULTS: SARS-CoV-2 was detected in all 21 known positive samples and none of the 16 negative samples. As few as approximately 5 viral copies were reliably detected. Since January 2021, many tissue types have been clinically tested. Of the 195 clinical specimens, the positivity rate was 35% with placenta and fetal tissue showing the highest percentage of positive cases. CONCLUSION: This sensitive FFPE-based assay has broad clinical utility with applications as diverse as pregnancy loss and evaluation of liver transplant rejection. This assay will aid in understanding atypical presentations of COVID-19 as well as long-term sequelae.


Subject(s)
COVID-19 , RNA, Viral , Real-Time Polymerase Chain Reaction , SARS-CoV-2 , COVID-19/diagnosis , Formaldehyde , Humans , Paraffin Embedding , RNA, Viral/isolation & purification , SARS-CoV-2/genetics
9.
Lancet ; 399(10326): 757-768, 2022 02 19.
Article in English | MEDLINE | ID: covidwho-1747476

ABSTRACT

Diagnostics have proven to be crucial to the COVID-19 pandemic response. There are three major methods for the detection of SARS-CoV-2 infection and their role has evolved during the course of the pandemic. Molecular tests such as PCR are highly sensitive and specific at detecting viral RNA, and are recommended by WHO for confirming diagnosis in individuals who are symptomatic and for activating public health measures. Antigen rapid detection tests detect viral proteins and, although they are less sensitive than molecular tests, have the advantages of being easier to do, giving a faster time to result, of being lower cost, and able to detect infection in those who are most likely to be at risk of transmitting the virus to others. Antigen rapid detection tests can be used as a public health tool for screening individuals at enhanced risk of infection, to protect people who are clinically vulnerable, to ensure safe travel and the resumption of schooling and social activities, and to enable economic recovery. With vaccine roll-out, antibody tests (which detect the host's response to infection or vaccination) can be useful surveillance tools to inform public policy, but should not be used to provide proof of immunity, as the correlates of protection remain unclear. All three types of COVID-19 test continue to have a crucial role in the transition from pandemic response to pandemic control.


Subject(s)
COVID-19 Testing/trends , COVID-19/diagnosis , Communicable Disease Control/organization & administration , Mass Screening/organization & administration , Pandemics/prevention & control , Antibodies, Viral/blood , Antigens, Viral/isolation & purification , COVID-19/epidemiology , COVID-19/transmission , COVID-19/virology , COVID-19 Testing/methods , COVID-19 Vaccines/administration & dosage , Communicable Disease Control/methods , Communicable Disease Control/trends , Humans , Mass Screening/trends , RNA, Viral/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification
10.
Viruses ; 12(6)2020 06 08.
Article in English | MEDLINE | ID: covidwho-1726020

ABSTRACT

Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 minute contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.


Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections/virology , Pneumonia, Viral/virology , Virus Inactivation/drug effects , Animals , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Betacoronavirus/physiology , COVID-19 , Cell Culture Techniques/methods , Chlorocebus aethiops , Containment of Biohazards/methods , Containment of Biohazards/standards , Humans , Nasopharynx/virology , Pandemics , RNA, Viral/isolation & purification , SARS-CoV-2 , Specimen Handling/methods , Vero Cells , Viral Load/methods
11.
Sci Rep ; 12(1): 2883, 2022 02 21.
Article in English | MEDLINE | ID: covidwho-1707349

ABSTRACT

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as 'direct to PCR' assays that do not involve RNA extraction or chemical neutralisation methods.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Containment of Biohazards/methods , Hot Temperature , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Specimen Handling/methods , Virus Inactivation , Animals , COVID-19/virology , Cell Line , Humans , Mice , Murine hepatitis virus/genetics , RNA, Viral/genetics , RNA, Viral/isolation & purification , Sensitivity and Specificity
12.
CMAJ Open ; 10(1): E74-E81, 2022.
Article in English | MEDLINE | ID: covidwho-1703594

ABSTRACT

BACKGROUND: During the first wave of the COVID-19 pandemic, a substantial number of Quebec hospitals were hit by hospital-acquired (HA) SARS-CoV-2 infections. Our objective was to assess whether mortality is higher in HA cases than in non-hospital-acquired (NHA) cases and determine the prevalence of HA-SARS-CoV-2 infection in our hospital. METHODS: This retrospective single-centre cohort study included all adults (≥ 18 yr) who had COVID-19, admitted to Hôpital Maisonneuve-Rosemont (Montréal, Canada) from Mar. 1 to June 30, 2020. We collected data on demographic characteristics, comorbidities, treatment, admission to the intensive care unit (ICU) and mechanical ventilation requirements from electronic health records. We adjudicated hospital acquisition based on the timing of symptom onset, and polymerase chain reaction testing for and exposures to SARS-CoV-2. To evaluate the association between HA-SARS-CoV-2 infection and in-hospital mortality, we computed a multivariable logistic regression analysis including known risk factors for death in patients with COVID-19 as covariates. RESULTS: Among 697 patients with SARS-CoV-2 infection, 253 (36.3%) were classified as HA. The mortality rate was higher in the HA group than in the NHA group (38.2% v. 26.4%, p = 0.001), while the rates of ICU admission (8.3% v. 19.1%, p = 0.001) and requirement for mechanical ventilation (3.6% v. 13.0%, p = 0.001) were lower. Multivariable logistic regression analysis showed that HA-SARS-CoV-2 infection in patients younger than 75 years is an independent risk factor for death (odds ratio 2.78, 95% confidence interval 1.44-5.38). INTERPRETATION: Our results show that HA-SARS-CoV-2 infection in younger patients was associated with higher mortality. Future studies need to evaluate relevant patient-centred long-term outcomes in this population.


Subject(s)
COVID-19/mortality , Iatrogenic Disease/epidemiology , Adolescent , Adult , Age Factors , Aged , Aged, 80 and over , COVID-19/diagnosis , COVID-19/therapy , COVID-19/virology , COVID-19 Nucleic Acid Testing , Female , Hospital Mortality , Humans , Intensive Care Units/statistics & numerical data , Male , Middle Aged , Pandemics/statistics & numerical data , Quebec/epidemiology , RNA, Viral/isolation & purification , Respiration, Artificial/statistics & numerical data , Retrospective Studies , Risk Assessment , Risk Factors , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Treatment Outcome , Young Adult
13.
Nat Commun ; 13(1): 968, 2022 02 18.
Article in English | MEDLINE | ID: covidwho-1705624

ABSTRACT

DNA/RNA-gold nanoparticle (DNA/RNA-AuNP) nanoprobes have been widely employed for nanobiotechnology applications. Here, we discover that both thiolated and non-thiolated DNA/RNA can be efficiently attached to AuNPs to achieve high-stable spherical nucleic acid (SNA) within minutes under a domestic microwave (MW)-assisted heating-dry circumstance. Further studies show that for non-thiolated DNA/RNA the conjugation is poly (T/U) tag dependent. Spectroscopy, test strip hybridization, and loading counting experiments indicate that low-affinity poly (T/U) tag mediates the formation of a standing-up conformation, which is distributed in the outer layer of SNA structure. In further application studies, CRISPR/Cas9-sgRNA (136 bp), SARS-CoV-2 RNA fragment (1278 bp), and rolling circle amplification (RCA) DNA products (over 1000 bp) can be successfully attached on AuNPs, which overcomes the routine methods in long-chain nucleic acid-AuNP conjugation, exhibiting great promise in biosensing and nucleic acids delivery applications. Current heating-dry strategy has improved traditional DNA/RNA-AuNP conjugation methods in simplicity, rapidity, cost, and universality.


Subject(s)
Biosensing Techniques/methods , Gold/chemistry , Metal Nanoparticles/chemistry , Biotechnology/methods , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing/methods , DNA/chemistry , Heating/methods , Humans , Limit of Detection , Microwaves , Nanomedicine/methods , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/isolation & purification , SARS-CoV-2/genetics
14.
Epidemiol Infect ; 150: e18, 2021 09 15.
Article in English | MEDLINE | ID: covidwho-1665657

ABSTRACT

Nosocomial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreaks among health care workers have been scarcely reported so far. This report presents the results of an epidemiologic and molecular investigation of a SARS-CoV-2 outbreak among laundromat facility workers in a large tertiary centre in Israel. Following the first three reported cases of SARS-CoV-2 among laundromat workers, all 49 laundromat personnel were screened by qRT-PCR tests using naso- and oropharingeal swabs. Epidemiologic investigations included questionnaires, interviews and observations of the laundromat facility. Eleven viral RNA samples were then sequenced, and a phylogenetic analysis was performed using MEGAX.The integrated investigation defined three genetic clusters and helped identify the index cases and the assumed routes of transmission. It was then deduced that shared commute and public showers played a role in SARS-CoV-2 transmission in this outbreak, in addition to improper PPE use and social gatherings (such as social eating and drinking). In this study, we present an integrated epidemiologic and molecular investigation may help detect the routes of SARS-CoV-2 transmission, emphasising such routes that are less frequently discussed. Our work reinforces the notion that person-to-person transmission is more likely to cause infections than environmental contamination (e.g. from handling dirty laundry).


Subject(s)
COVID-19/epidemiology , Disease Outbreaks , Laundry Service, Hospital , SARS-CoV-2 , Adult , COVID-19/transmission , Cohort Studies , Contact Tracing , Cross Infection/epidemiology , Cross Infection/transmission , Cross Infection/virology , Disease Outbreaks/statistics & numerical data , Female , Humans , Israel/epidemiology , Male , Middle Aged , Phylogeny , RNA, Viral/chemistry , RNA, Viral/isolation & purification , SARS-CoV-2/classification , SARS-CoV-2/genetics
15.
PLoS One ; 17(1): e0262178, 2022.
Article in English | MEDLINE | ID: covidwho-1637832

ABSTRACT

BACKGROUND: COVID-19 is an ongoing public health pandemic regardless of the countless efforts made by various actors. Quality diagnostic tests are important for early detection and control. Notably, several commercially available one step RT-PCR based assays have been recommended by the WHO. Yet, their analytic and diagnostic performances have not been well documented in resource-limited settings. Hence, this study aimed to evaluate the diagnostic sensitivities and specificities of three commercially available one step reverse transcriptase-polymerase chain reaction (RT-PCR) assays in Ethiopia in clinical setting. METHODS: A cross-sectional study was conducted from April to June, 2021 on 279 respiratory swabs originating from community surveillance, contact cases and suspect cases. RNA was extracted using manual extraction method. Master-mix preparation, amplification and result interpretation was done as per the respective manufacturer. Agreements between RT-PCRs were analyzed using kappa values. Bayesian latent class models (BLCM) were fitted to obtain reliable estimates of diagnostic sensitivities, specificities of the three assays and prevalence in the absence of a true gold standard. RESULTS: Among the 279 respiratory samples, 50(18%), 59(21.2%), and 69(24.7%) were tested positive by TIB, Da An, and BGI assays, respectively. Moderate to substantial level of agreement was reported among the three assays with kappa value between 0 .55 and 0.72. Based on the BLCM relatively high specificities (95% CI) of 0.991(0.973-1.000), 0.961(0.930-0.991) and 0.916(0.875-0.952) and considerably lower sensitivities with 0.813(0.658-0.938), 0.836(0.712-0.940) and 0.810(0.687-0.920) for TIB MOLBIOL, Da An and BGI respectively were found. CONCLUSIONS: While all the three RT-PCR assays displayed comparable sensitivities, the specificities of TIB MOLBIOL and Da An were considerably higher than BGI. These results help adjust the apparent prevalence determined by the three RT-PCRs and thus support public health decisions in resource limited settings and consider alternatives as per their prioritization matrix.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/epidemiology , Diagnostic Tests, Routine/methods , Pandemics/prevention & control , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Bayes Theorem , COVID-19/virology , Child , Cross-Sectional Studies , Ethiopia/epidemiology , False Positive Reactions , Female , Humans , Male , Middle Aged , Molecular Diagnostic Techniques/methods , RNA, Viral/genetics , RNA, Viral/isolation & purification , Sensitivity and Specificity , Young Adult
16.
PLoS One ; 17(1): e0262170, 2022.
Article in English | MEDLINE | ID: covidwho-1637228

ABSTRACT

The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike gene. Monitoring the S gene mutations is crucial for successful controlling measures and detecting variants that can evade vaccine immunity. Even after the costs reduction resulting from the pandemic, the new generation sequencing methodologies remain unavailable to a large number of scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S gene, this work describes a new feasible protocol for complete nucleotide sequencing of the S gene using the Sanger technique. Such a methodology could be easily adopted by any laboratory with experience in sequencing, adding to effective surveillance of SARS-CoV-2 spreading and evolution.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/epidemiology , Genes, Viral , Pandemics/prevention & control , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sequence Analysis, RNA/methods , Spike Glycoprotein, Coronavirus/genetics , Base Sequence , Brazil/epidemiology , COVID-19/virology , Diagnostic Tests, Routine/methods , Electrophoresis, Agar Gel/methods , Epidemiological Monitoring , Humans , Mutation , RNA, Viral/genetics , RNA, Viral/isolation & purification
17.
Dis Markers ; 2022: 6478434, 2022.
Article in English | MEDLINE | ID: covidwho-1622116

ABSTRACT

BACKGROUND: Since the beginning of the pandemic, clinicians and researchers have been searching for alternative tests to improve the screening and diagnosis of the SARS-CoV-2 infection. Currently, the gold standard for virus identification is the nasopharyngeal (NP) swab. Saliva samples, however, offer clear, practical, and logistical advantages but due to a lack of collection, transport, and storage solutions, high-throughput saliva-based laboratory tests are difficult to scale up as a screening or diagnostic tool. With this study, we aimed to validate an intralaboratory molecular detection method for SARS-CoV-2 on saliva samples collected in a new storage saline solution, comparing the results to NP swabs to determine the difference in sensitivity between the two tests. METHODS: In this study, 156 patients (cases) and 1005 asymptomatic subjects (controls) were enrolled and tested simultaneously for the detection of the SARS-CoV-2 viral genome by RT-PCR on both NP swab and saliva samples. Saliva samples were collected in a preservative and inhibiting saline solution (Biofarma Srl). Internal method validation was performed to standardize the entire workflow for saliva samples. RESULTS: The identification of SARS-CoV-2 conducted on saliva samples showed a clinical sensitivity of 95.1% and specificity of 97.8% compared to NP swabs. The positive predictive value (PPV) was 81% while the negative predictive value (NPV) was 99.5%. Test concordance was 97.6% (Cohen's Kappa = 0.86; 95% CI 0.81-0.91). The LoD of the test was 5 viral copies for both samples. CONCLUSIONS: RT-PCR assays conducted on a stored saliva sample achieved similar performance to those on NP swabs, and this may provide a very effective tool for population screening and diagnosis. Collection of saliva in a stabilizing solution makes the test more convenient and widely available; furthermore, the denaturing properties of the solution reduce the infective risks belonging to sample manipulation.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Saliva/virology , Adult , Aged , Case-Control Studies , Humans , Middle Aged , Nasopharynx/virology , Predictive Value of Tests , RNA, Viral/isolation & purification , SARS-CoV-2/genetics , Specimen Handling/methods
18.
Sci Total Environ ; 821: 152790, 2022 May 15.
Article in English | MEDLINE | ID: covidwho-1612001

ABSTRACT

Affordably tracking the transmission of respiratory infectious diseases in urban transport infrastructures can inform individuals about potential exposure to diseases and guide public policymakers to prepare timely responses based on geographical transmission in different areas in the city. Towards that end, we designed and tested a method to detect SARS-CoV-2 RNA in the air filters of public buses, revealing that air filters could be used as passive fabric sensors for the detection of viral presence. We placed and retrieved filters in the existing HVAC systems of public buses to test for the presence of trapped SARS-CoV-2 RNA using phenol-chloroform extraction and RT-qPCR. SARS-CoV-2 RNA was detected in 14% (5/37) of public bus filters tested in Seattle, Washington, from August 2020 to March 2021. These results indicate that this sensing system is feasible and that, if scaled, this method could provide a unique lens into the geographically relevant transmission of SARS-CoV-2 through public transit rider vectors, pooling samples of riders over time in a passive manner without installing any additional systems on transit vehicles.


Subject(s)
Motor Vehicles , RNA, Viral/isolation & purification , SARS-CoV-2 , Transportation , COVID-19 , Environmental Monitoring , Humans , SARS-CoV-2/isolation & purification , Washington
19.
Yakugaku Zasshi ; 142(1): 11-15, 2022.
Article in Japanese | MEDLINE | ID: covidwho-1609123

ABSTRACT

The polio eradication program, launched in 1988, has successfully decreased the number of poliomyelitis patients worldwide. However, in areas with immunization gaps where oral polio vaccine coverage has dropped, outbreaks of more virulent vaccine-derived polioviruses (VDPVs) have become a threat to public health. In Japan, inactivated polio vaccine replaced oral polio vaccine as the routine immunization in 2012. Polio environmental surveillance (ES) has been conducted nationwide since 2013 to efficiently monitor the wild type poliovirus or VDPV, which may be imported from overseas. ES may also be utilized to detect other viruses in stool samples. We propose a method of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection based on the polio ES network, and establish a procedure to detect fragments of SARS-CoV-2 genome in wastewater solids. Our findings suggest that polio ES can be used to simultaneously monitor SARS-CoV-2 RNA fragments in sewage waters.


Subject(s)
Environmental Monitoring/methods , Poliovirus/isolation & purification , SARS-CoV-2/isolation & purification , Sewage/virology , Waste Water/virology , Disease Eradication , Humans , Japan , Poliovirus Vaccine, Inactivated , RNA, Viral/isolation & purification , SARS-CoV-2/genetics
20.
Microbiol Spectr ; 10(1): e0143821, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1608700

ABSTRACT

With the emergence and wide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), such as the Delta variant (B.1.617.2 lineage and AY sublineage), it is important to track VOCs for sourcing of transmission. Currently, whole-genome sequencing is commonly used for detecting VOCs, but this is limited by the high costs of reagents and sophisticated sequencers. In this study, common mutations in the genomes of SARS-CoV-2 VOCs were identified by analyzing more than 1 million SARS-CoV-2 genomes from public data. Among them, mutations C1709A (a change of C to A at position 1709) and C56G, respectively, were found in more than 99% of the genomes of Alpha and Delta variants and were specific to them. Then, a method using the amplification refractory mutation system combined with quantitative reverse transcription-PCR (ARMS-RT-qPCR) based on the two mutations was developed for identifying both VOCs. The assay can detect as little as 1 copy/µL of the VOCs, and the results for identifying Alpha and Delta variants in clinical samples by the ARMS-RT-qPCR assay showed 100% agreement with the results using sequencing-based methods. The whole assay can be completed in 2.5 h using commercial fluorescent PCR instruments. Therefore, the ARMS-RT-qPCR assay could be used for screening the two highly concerning variants Alpha and Delta by normal PCR laboratories in airports and in hospitals and other health-related organizations. Additionally, based on the unique mutations identified by the genomic analysis, similar molecular assays can be developed for rapid identification of other VOCs. IMPORTANCE The current stage of the pandemic, led by SARS-CoV-2 variants of concern (VOCs), underscores the necessity to develop a cost-effective and rapid molecular diagnosis assay to differentiate the VOCs. In this study, over 1 million SARS-CoV-2 genomic sequences of high quality from GISAID were analyzed and a network of the common mutations of the lineages was constructed. The conserved unique mutations specific for SARS-CoV-2 VOCs were found. Then, ARMS-RT-qPCR assays based on the two unique mutations of the Alpha and Delta variants were developed for the detection of the two VOCs. Application of the assay in clinical samples demonstrated that the current method is a convenient, cost-effective, and rapid way to screen the target SARS-CoV-2 VOCs.


Subject(s)
COVID-19/virology , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , Genome, Viral , High-Throughput Nucleotide Sequencing , Mutation , Nucleic Acid Amplification Techniques/trends , Pharynx/virology , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/classification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/classification , Spike Glycoprotein, Coronavirus/genetics
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