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2.
PLoS One ; 17(2): e0262784, 2022.
Article in English | MEDLINE | ID: covidwho-1793538

ABSTRACT

INTRODUCTION: Even if now we have available the weapon of vaccination against SARS-CoV-2, the patients with cancer remains a very frail population in which frequently the immunologic response to vaccination may be impaired. In this setting, the SARS-CoV-2 infection screening retains a great value. However, there are still limited data on the feasibility and efficacy of combined screening procedures to assess the prevalence of SARS-CoV-2 infection (including asymptomatic cases) in cancer outpatients undergoing antineoplastic therapy. PATIENTS AND RESULTS: From May 1, 2020, to June 15, 2020, during the first wave of SARS-CoV-2 pandemic, 860 consecutive patients, undergoing active anticancer therapy, were evaluated and tested for SARS-CoV-2 with a combined screening procedure, including a self-report questionnaire, a molecular nasopharyngeal swab (NPS) and a rapid serological immunoassay (for anti-SARS-CoV-2 IgG/IgM antibodies). The primary endpoint of the study was to estimate the prevalence of SARS-CoV-2 infection (including asymptomatic cases) in consecutive and unselected cancer outpatients by a combined screening modality. A total of 2955 SARS-CoV-2 NPS and 860 serological tests, in 475 patients with hematologic cancers and in 386 with solid tumors, were performed. A total of 112 (13%) patients self-reported symptoms potentially COVID-19 related. In 1/860 cases (< 1%) SARS-CoV-2 NPS was positive and in 14 cases (1.62%) the specific serological test was positive (overall prevalence of SARS-CoV-2 infection 1.62%). Of the 112 cases who declared symptoms potentially COVID-19-related, only 2.7% (3/112) were found SARS-CoV-2 positive. CONCLUSIONS: This is the largest study reporting the feasibility of a combined screening procedure (including triage, NPS and serologic test) to evaluate the prevalence of SARS-CoV-2 infection in cancer patients receiving active therapy, during the first epidemic wave and under the restrictive lockdown measures, in one of the active areas of the SARS-CoV-2 circulation. Lacking specific recommendations for the detection of asymptomatic SARS-CoV-2 cases, a combined diagnostic screening might be more effective to detect the exact prevalence of SARS-CoV-2 in neoplastic patient population. The prevalence can obviously change according to the territorial context, the entity of the restrictive measures adopted and the phase of the epidemic curve. However, its exact and real-time knowledge could be important to balance risks/benefits of oncologic treatments, avoiding (if the prevalence is low) the reduction of dose intensity or the selection of less intensive (but also less effective) anti-cancer therapies.


Subject(s)
COVID-19/diagnosis , Neoplasms/complications , Neoplasms/virology , Adult , Aged , Aged, 80 and over , Antibodies, Viral , Asymptomatic Infections/epidemiology , COVID-19/complications , Communicable Disease Control , Comorbidity , Diagnostic Screening Programs/trends , Female , Humans , Immunoglobulin G , Immunoglobulin M , Incidence , Italy/epidemiology , Male , Middle Aged , Nasopharynx/virology , Prevalence , SARS-CoV-2/pathogenicity , Serologic Tests
3.
J Med Microbiol ; 71(4)2022 Apr.
Article in English | MEDLINE | ID: covidwho-1788578

ABSTRACT

Saliva is an alternative sample material to nasopharyngeal swab in SARS-CoV-2 diagnostics. We investigated possible aspects to improve the reliability of SARS-CoV-2 detection from saliva. Saliva was collected from asymptomatic healthy subjects (n=133) and COVID-19 patients (n=9). SARS-CoV-2 detection was performed with quantitative reverse-transcriptase PCR (RT-qPCR) with two viral and one host target serving as an internal control. The use of internal control revealed that in the first RT-qPCR run 25-30 % of assays failed. The failure is associated with poor RNA quality. When the amount of RNA was cut down to half from the original amount, the performance of RT-qPCR was greatly enhanced (95 % of the assays succeeded). The quality of RNA was not affected by the use of different nucleic acid stabilizing buffers. Our study showed that saliva is suitable material for RT-qPCR based SARS-CoV-2 diagnostics, but the use of internal control is essential to distinguish the true negative samples from failed assays.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Nasopharynx , RNA, Viral/analysis , RNA, Viral/genetics , Reproducibility of Results , SARS-CoV-2/genetics , Saliva , Specimen Handling
4.
BMC Infect Dis ; 22(1): 221, 2022 Mar 04.
Article in English | MEDLINE | ID: covidwho-1779604

ABSTRACT

BACKGROUND: The nucleic acid amplification test (NAAT) and antigen test are approved diagnostic tests for COVID-19. In this study, we aimed to investigate the assay performance of two NAATs (Xpert Xpress SARS-CoV-2 and FilmArray Respiratory Panel) and a quantitative antigen test (Lumipulse). METHODS: One hundred and sixty-five nasopharyngeal swabs were subjected to Xpert, FilmArray, Lumipulse, and RT-qPCR assays. RESULTS: Of 165 samples, RT-qPCR showed 100 positives and 65 negatives. The Xpert had an overall agreement of 99.4% (95% confidence interval [CI]: 96.7-99.4%), sensitivity of 99% (95% CI: 96.8-99%), and specificity of 100% (95% CI: 96.6-100%). FilmArray had an overall agreement of 98.8% (95% CI: 95.9-98.8%), sensitivity of 98% (95% CI: 95.6-98%), and specificity of 100% (95% CI: 96.3-100%). Lumipulse had an overall agreement of 95.5% (95% CI: 91.8-95.5%), sensitivity of 92.3% (95% CI: 89.2-92.3%), and specificity of 100% (95% CI: 95.5-100%). The κ coefficient showed excellent agreement between each test and RT-qPCR. There was a high correlation between Xpert Ct values, RT-qPCR Ct values, viral loads and antigen level. CONCLUSIONS: Xpert Xpress and FilmArray Respiratory Panel exhibited an equivalent performance. The Lumipulse antigen test was slightly less sensitive than the NAATs, but showed high assay performance except for samples with low viral load. The Xpert Xpress, FilmArray Respiratory Panel and Lumipulse antigen tests offer rapid sample-to-answer data, allowing random access detection on automated devices.


Subject(s)
COVID-19 , COVID-19/diagnosis , COVID-19 Testing , Humans , Nasopharynx , SARS-CoV-2 , Sensitivity and Specificity
5.
J Med Virol ; 94(5): 2284-2289, 2022 05.
Article in English | MEDLINE | ID: covidwho-1777584

ABSTRACT

Remdesivir is a broad-spectrum antiviral agent able to inhibit the RNA polymerase of SARS-CoV-2. At present, studies focusing on the effect of remdesivir on viral load (VL) are few and with contrasting results. Aim of the present study was to evaluate the effect of remdesivir on SARS-CoV-2 VL from nasopharyngeal swabs (cycle threshold criterion) in a sample of patients treated with the drug, compared with patients who did not receive the antiviral treatment. This retrospective analysis evaluated patients with (1) real-time polymerase chain reaction (RT-PCR) confirmed COVID-19 diagnosis and (2) availability of at least two positive nasopharyngeal swabs analysed with the same analytic platform (ORF target gene, Ingenius ELITe, ELITechGroup, Puteaux, France). Upper respiratory specimens from nasopharyngeal swabs were collected at admission (T0) and 7-14 days after treatment, upon clinical decision. A total of 27 patients treated with remdesivir (Group A) met the inclusion criteria and were compared with 18 patients (Group B) treated with standard care, matched for baseline clinical characteristics. At baseline, both remdesivir-treated and nontreated patients showed comparable VLs (21.73 ± 6.81 vs. 19.27 ± 5.24, p = 0.348). At the second swab, remdesivir-treated patients showed a steeper VL reduction with respect to controls (34.28 ± 7.73 vs. 27.22 ± 3.92; p < 0.001). Longitudinal linear model estimated a mean decrease in cycle threshold equal to 0.61 (SE: 0.09) per day in remdesivir-treated versus 0.33 (SE: 0.10) per day in remdesivir nontreated patients (p for heterogeneity = 0.045). The present study shows that the administration of remdesivir in hospitalized COVID-19 patients significantly reduces the VL on nasopharyngeal swabs.


Subject(s)
COVID-19 , SARS-CoV-2 , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , COVID-19/diagnosis , COVID-19/drug therapy , COVID-19 Testing , Case-Control Studies , Humans , Nasopharynx , Retrospective Studies , Viral Load
6.
Sci Rep ; 12(1): 5369, 2022 Mar 30.
Article in English | MEDLINE | ID: covidwho-1768848

ABSTRACT

The COVID-19 pandemic requires sensitive detection of the SARS-CoV-2 virus from samples to ensure accurate detection of infected patients, an essential component of effective national track and trace programs. Due to the scaling challenges of large sample numbers, sample pooling is an attractive solution to reduce both extraction and amplification reagent costs, if high sensitivity can be maintained. We demonstrate that the Erba Molecular ErbaMDx SARS-CoV-2 RT-PCR Kit (EM kit) delivers high sensitivity, achieving analytical detection of 5 copies/reaction SARS-CoV-2 genomic RNA, and 200 copies/mL SARS-CoV-2 inactivated virus spiked into nasopharyngeal swab (NP) samples and extracted through workflow. Furthermore, the EM Kit demonstrates high sensitivity in both pooled (1 in 5) and non-pooled NP samples when compared to an FDA Emergency Use Authorization approved assay, following published FDA guidelines. These findings demonstrate that the EM Kit is suitable for sample pooling, with minimal impact on assay performance. As the COVID-19 pandemic progresses, high sensitivity assays such as the EM Kit will have an important role in ensuring high throughput and sensitive testing using pooled samples can be maintained, delivering the most cost-effective sample extraction and amplification option for national test and trace programs.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Nasopharynx , Pandemics , RNA, Viral/analysis , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity
7.
Microb Pathog ; 165: 105506, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1763898

ABSTRACT

Since its first appearance, the SARS-CoV-2 has spread rapidly in the human population, reaching the pandemic scale with >280 million confirmed infections and more than 5 million deaths to date (https://covid19.who.int/). These data justify the urgent need to enhance our understanding of SARS-CoV-2 effects in the respiratory system, including those linked to co-infections. The principal aim of our study is to investigate existing correlations in the nasopharynx between the bacterial community, potential pathogens, and SARS-CoV-2 infection. The main aim of this study was to provide evidence pointing to possible relationships between components of the bacterial community and SARS-CoV-2 in the nasopharynx. Meta-transcriptomic profiling of the nasopharyngeal microbial community was carried out in 89 SARS-Cov-2 positive subjects from the Campania Region in Italy. To this end, RNA extracted from nasopharyngeal swabs collected at different times during the initial phases of the pandemic was analyzed by Next-Generation Sequencing (NGS). Results show a consistently high presence of members of the Proteobacteria (41.85%), Firmicutes (28.54%), and Actinobacteria (16.10%) phyla, and an inverted correlation between the host microbiome, co-infectious bacteria, and super-potential pathogens such as Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Neisseria gonorrhoeae. In depth characterization of microbiota composition in the nasopharynx can provide clues to understand its potential contribution to the clinical phenotype of Covid-19, clarifying the interaction between SARS-Cov-2 and the bacterial flora of the host, and highlighting its dysbiosis and the presence of pathogens that could affect the patient's disease progression and outcome.


Subject(s)
COVID-19 , Coinfection , Microbiota , Bacteria/genetics , Coinfection/epidemiology , High-Throughput Nucleotide Sequencing , Humans , Italy/epidemiology , Microbiota/genetics , Nasopharynx/microbiology , Pandemics , SARS-CoV-2/genetics
9.
Rev Fac Cien Med Univ Nac Cordoba ; 79(1): 91-94, 2022 03 07.
Article in Spanish | MEDLINE | ID: covidwho-1754226

ABSTRACT

Introduction: In relation to the nasopharynx swab samples, necessary to identify SARS-CoV-2, various problems have been reported, such as: delays in viral identification, high percentages of false negative PCR and viral presence in asymptomatic patients. This, and because the swab is performed without intranasal vision, suggests the need to improve the process of obtaining the nasopharyngeal swab sample, the most important step in viral identification. Description: Because nasal endoscopy is a procedure that provides direct visualization of intranasal structures; We propose its use to support laboratory personnel in obtaining the nasopharyngeal swab, allowing to improve the quality of the sample, with greater possibilities of early viral identification in COVID-19 patients. In addition, performing endoscopy through a posterior approach would reduce the risk of contagion from the personnel who perform it. Conclusion: We propose the use of posterior nasal endoscopy as support in obtaining the nasopharyngeal swab sample, to improve the identification of SARS-CoV-2. Its realization by means of the posterior approach, would avoid the contagion of the personnel who perform it.


Introducción: En relación a las muestras de hisopado de nasofaringe, necesarias para identificar al SARS-CoV-2, se han reportado diversos problemas como: retrasos en la identificación viral, elevados porcentajes de falsos negativos de PCR y presencia viral en pacientes asintomáticos. Lo anterior, y debido a que el hisopado se realiza sin visión intranasal, sugiere la necesidad de mejorar el proceso de obtención de la muestra del hisopado nasofaríngeo, el paso más importante en la identificación viral. Descripción: Debido a que la endoscopía nasal es un procedimiento que proporciona visualización directa de las estructuras intranasales; proponemos su utilización en apoyo al personal de laboratorio en la obtención del hisopado nasofaríngeo, permitiendo mejorar la calidad de la muestra, con mayores posibilidades de identificación viral precoz en pacientes COVID-19. Además, la realización de la endoscopía mediante un abordaje posterior, disminuiría el riesgo de contagio del personal que lo realiza. Conclusión: Proponemos el uso de la endoscopía nasal posterior como apoyo en la obtención de la muestra del hisopado nasofaríngeo, para mejorar la identificación del SARS-CoV-2. Su realización mediante el abordaje posterior, evitaría el contagio del personal que lo realiza.


Subject(s)
COVID-19 , COVID-19/diagnosis , Endoscopy , Humans , Nasopharynx , SARS-CoV-2 , Specimen Handling/methods
10.
J Korean Med Sci ; 37(11): e88, 2022 Mar 21.
Article in English | MEDLINE | ID: covidwho-1753355

ABSTRACT

Nasopharyngeal swabs have been widely to prevent the spread of coronavirus disease 2019 (COVID-19). Nasopharyngeal COVID-19 testing is a generally safe and well-tolerated procedure, but numerous complications have been reported in the media. Therefore, the present study aimed to review and document adverse events and suggest procedural references to minimize preventable but often underestimated risks. A total of 27 articles were selected for the review of 842 related documents in PubMed, Embase, and KoreaMed. The complications related to nasopharyngeal COVID-19 testing were reported to be rarely happened, ranging from 0.0012 to 0.026%. Frequently documented adverse events were retained swabs, epistaxis, and cerebrospinal fluid leakage, often associated with high-risk factors, including severe septal deviations, pre-existing skull base defects, and previous sinus or transsphenoidal pituitary surgery. Appropriate techniques based on sufficient anatomical knowledge are mandatory for clinicians to perform nasopharyngeal COVID-19 testing. The nasal floor can be predicted by the line between the nostril and external ear canal. For safe testing, the angle of swab insertion in the nasal passage should remain within 30° of the nasal floor. The swab was gently inserted along the nasal septum just above the nasal floor to the nasopharynx and remained on the nasopharynx for several seconds before removal. Forceful insertion should be attempted, and alternative examinations should be considered, especially in vulnerable patients. In conclusion, patients and clinicians should be aware of rare but possible complications and associated high-risk factors. The suggested procedural pearls enable more comfortable and safe nasopharyngeal COVID-19 testing for both clinicians and patients.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Nasopharynx/virology , SARS-CoV-2/isolation & purification , Specimen Handling/adverse effects , Humans , Nasal Cavity/anatomy & histology , Nasal Cavity/virology , Nasopharynx/anatomy & histology , Specimen Handling/methods
11.
J Transl Med ; 20(1): 134, 2022 03 18.
Article in English | MEDLINE | ID: covidwho-1745446

ABSTRACT

BACKGROUND: A thorough understanding of a patient's inflammatory response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is crucial to discerning the associated, underlying immunological processes and to the selection and implementation of treatment strategies. Defining peripheral blood biomarkers relevant to SARS-CoV-2 infection is fundamental to detecting and monitoring this systemic disease. This safety-focused study aims to monitor and characterize the immune response to SARS-CoV-2 infection via analysis of peripheral blood and nasopharyngeal swab samples obtained from patients hospitalized with Coronavirus disease 2019 (COVID-19), in the presence or absence of bamlanivimab treatment. METHODS: 23 patients hospitalized with COVID-19 were randomized to receive a single dose of the neutralizing monoclonal antibody, bamlanivimab (700 mg, 2800 mg or 7000 mg) or placebo, at study initiation (Clinical Trial; NCT04411628). Serum samples and nasopharyngeal swabs were collected at multiple time points over 1 month. A Proximity Extension Array was used to detect inflammatory profiles from protein biomarkers in the serum of hospitalized COVID-19 patients relative to age/sex-matched healthy controls. RNA sequencing was performed on nasopharyngeal swabs. A Luminex serology assay and Elecsys® Anti-SARS-CoV-2 immunoassay were used to detect endogenous antibody formation and to monitor seroconversion in each cohort over time. A mixed model for repeated measures approach was used to analyze changes in serology and serum proteins over time. RESULTS: Levels of IL-6, CXCL10, CXCL11, IFNγ and MCP-3 were > fourfold higher in the serum of patients with COVID-19 versus healthy controls and linked with observations of inflammatory and viral-induced interferon response genes detected in nasopharyngeal swab samples from the same patients. While IgA and IgM titers peaked around 7 days post-dose, IgG titers remained high, even after 28 days. Changes in biomarkers over time were not significantly different between the bamlanivimab and placebo groups. CONCLUSIONS: Similarities observed between nasopharyngeal gene expression patterns and peripheral blood biomarker profiles reveal a connection between the circulation and processes in the nasopharyngeal cavity, reinforcing the potential utility of systemic blood biomarker profiling for therapeutic monitoring of patient response. Serological antibody responses in patients correlated closely with reductions in the COVID-19 inflammatory protein biomarker signature. Bamlanivimab did not affect the biomarker dynamics in this hospitalized patient population.


Subject(s)
COVID-19 , Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Neutralizing , Antibodies, Viral , Biomarkers , COVID-19/drug therapy , Gene Expression , Humans , Nasopharynx , SARS-CoV-2
12.
PLoS One ; 17(3): e0264085, 2022.
Article in English | MEDLINE | ID: covidwho-1736504

ABSTRACT

Self-collected specimens can expand access to SARS-CoV-2 testing. At a large inner-city hospital 1,082 participants self-collected saliva and anterior nasal swab (ANS) samples before healthcare workers collected nasopharyngeal swab (NPS) samples on the same day. To characterize patient preferences for self-collection, this investigation explored ability, comfort, and ease of ANS and saliva self-collection for SARS-CoV-2 testing along with associated patient characteristics, including medical history and symptoms of COVID-19. With nearly all participants successfully submitting a specimen, favorable ratings from most participants (at least >79% in ease and comfort), and equivocal preference between saliva and ANS, self-collection is a viable SARS-CoV-2 testing option.


Subject(s)
COVID-19/diagnosis , Specimen Handling/methods , Adolescent , Adult , COVID-19/virology , COVID-19 Testing , Female , Georgia , Humans , Male , Middle Aged , Nasopharynx/virology , RNA, Viral/analysis , RNA, Viral/metabolism , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Saliva/virology , Young Adult
13.
Sci Rep ; 12(1): 4132, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735286

ABSTRACT

This paper presents a deep learning-driven portable, accurate, low-cost, and easy-to-use device to perform Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) to facilitate rapid detection of COVID-19. The 3D-printed device-powered using only a 5 Volt AC-DC adapter-can perform 16 simultaneous RT-LAMP reactions and can be used multiple times. Moreover, the experimental protocol is devised to obviate the need for separate, expensive equipment for RNA extraction in addition to eliminating sample evaporation. The entire process from sample preparation to the qualitative assessment of the LAMP amplification takes only 45 min (10 min for pre-heating and 35 min for RT-LAMP reactions). The completion of the amplification reaction yields a fuchsia color for the negative samples and either a yellow or orange color for the positive samples, based on a pH indicator dye. The device is coupled with a novel deep learning system that automatically analyzes the amplification results and pays attention to the pH indicator dye to screen the COVID-19 subjects. The proposed device has been rigorously tested on 250 RT-LAMP clinical samples, where it achieved an overall specificity and sensitivity of 0.9666 and 0.9722, respectively with a recall of 0.9892 for Ct < 30. Also, the proposed system can be widely used as an accurate, sensitive, rapid, and portable tool to detect COVID-19 in settings where access to a lab is difficult, or the results are urgently required.


Subject(s)
COVID-19/diagnosis , Deep Learning , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , Area Under Curve , COVID-19 Testing , Coloring Agents/chemistry , Humans , Molecular Diagnostic Techniques/instrumentation , Nasopharynx/virology , Nucleic Acid Amplification Techniques/instrumentation , Point-of-Care Systems , Printing, Three-Dimensional , RNA, Viral/analysis , RNA, Viral/metabolism , ROC Curve , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
14.
Sci Rep ; 12(1): 3706, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735275

ABSTRACT

Scaling up SARS-CoV-2 testing and tracing continues to be plagued with the limitation of the sample collection method, which requires trained healthcare workers to perform and causes discomfort to the patients. In response, we assessed the performance and user preference of gargle specimens for qRT-PCR-based detection of SARS-CoV-2 in Indonesia. Inpatients who had recently been diagnosed with COVID-19 and outpatients who were about to perform qRT-PCR testing were asked to provide nasopharyngeal and oropharyngeal (NPOP) swabs and self-collected gargle specimens. We demonstrated that self-collected gargle specimens can be an alternative specimen to detect SARS-CoV-2 and the viral RNA remained stable for 31 days at room temperature storage. The developed method was validated for use on multiple RNA extraction kits and commercially available COVID-19 RT-PCR kits. Our developed method achieved a sensitivity of 91.38% when compared to paired NPOP swab specimens (Ct < 35), with 97.10% of patients preferring the self-collected gargle method.


Subject(s)
COVID-19/diagnosis , Saliva/virology , Specimen Handling/methods , COVID-19/virology , Humans , Mouthwashes/chemistry , Nasopharynx/virology , Oropharynx/virology , RNA, Viral/analysis , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
15.
Sci Rep ; 12(1): 3775, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735272

ABSTRACT

Loop-mediated isothermal amplification is known for its high sensitivity, specificity and tolerance to inhibiting-substances. In this work, we developed a device for performing real-time colorimetric LAMP combining the accuracy of lab-based quantitative analysis with the simplicity of point-of-care testing. The device innovation lies on the use of a plastic tube anchored vertically on a hot surface while the side walls are exposed to a mini camera able to take snapshots of the colour change in real time during LAMP amplification. Competitive features are the rapid analysis (< 30 min), quantification over 9 log-units, crude sample-compatibility (saliva, tissue, swabs), low detection limit (< 5 copies/reaction), smartphone-operation, fast prototyping (3D-printing) and ability to select the dye of interest (Phenol red, HNB). The device's clinical utility is demonstrated in cancer mutations-analysis during the detection of 0.01% of BRAF-V600E-to-wild-type molecules from tissue samples and COVID-19 testing with 97% (Ct < 36.8) and 98% (Ct < 30) sensitivity when using extracted RNA and nasopharyngeal-swabs, respectively. The device high technology-readiness-level makes it a suitable platform for performing any colorimetric LAMP assay; moreover, its simple and inexpensive fabrication holds promise for fast deployment and application in global diagnostics.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , Colorimetry , Humans , Limit of Detection , Molecular Diagnostic Techniques , Nasopharynx/virology , Neoplasms/diagnosis , Neoplasms/genetics , Neoplasms/pathology , Nucleic Acid Amplification Techniques , Point-of-Care Testing , Proto-Oncogene Proteins B-raf/genetics , RNA, Viral/analysis , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Smartphone
16.
Sci Rep ; 12(1): 3480, 2022 03 03.
Article in English | MEDLINE | ID: covidwho-1730307

ABSTRACT

The COVID-19 pandemic has resulted in significant diversion of human and material resources to COVID-19 diagnostics, to the extent that influenza viruses and co-infection in COVID-19 patients remains undocumented and pose serious public-health consequences. We optimized and validated a highly sensitive RT-PCR based multiplex-assay for the detection of SARS-CoV-2, influenza A and B viruses in a single-test. This study evaluated clinical specimens (n = 1411), 1019 saliva and 392 nasopharyngeal swab (NPS), tested using two-assays: FDA-EUA approved SARS-CoV-2 assay that targets N and ORF1ab gene, and the PKamp-RT-PCR based assay that targets SARS-CoV-2, influenza viruses A and B. Of the 1019 saliva samples, 17.0% (174/1019) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [91.9% (160/174) vs. 87.9% (153/174)], respectively. Of the 392 NPS samples, 10.4% (41/392) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [97.5% (40/41) vs. 92.1% (39/41)], respectively. This study presents clinical validation of a multiplex-PCR assay for testing SARS-CoV-2, influenza A and B viruses, using NPS and saliva samples, and demonstrates the feasibility of implementing the assay without disrupting the existing laboratory workflow.


Subject(s)
Influenza A virus/isolation & purification , Influenza B virus/isolation & purification , Multiplex Polymerase Chain Reaction/methods , Nasopharynx/virology , SARS-CoV-2/isolation & purification , Saliva/virology , Humans , Limit of Detection , Reproducibility of Results
17.
Viruses ; 12(6)2020 06 10.
Article in English | MEDLINE | ID: covidwho-1726022

ABSTRACT

There is currently debate about human coronavirus (HCoV) seasonality and pathogenicity, as epidemiological data are scarce. Here, we provide epidemiological and clinical features of HCoV patients with acute respiratory infection (ARI) examined in primary care general practice. We also describe HCoV seasonality over six influenza surveillance seasons (week 40 to 15 of each season) from the period 2014/2015 to 2019/2020 in Corsica (France). A sample of patients of all ages presenting for consultation for influenza-like illness (ILI) or ARI was included by physicians of the French Sentinelles Network during this period. Nasopharyngeal samples were tested for the presence of 21 respiratory pathogens by real-time RT-PCR. Among the 1389 ILI/ARI patients, 105 were positive for at least one HCoV (7.5%). On an annual basis, HCoVs circulated from week 48 (November) to weeks 14-15 (May) and peaked in week 6 (February). Overall, among the HCoV-positive patients detected in this study, HCoV-OC43 was the most commonly detected virus, followed by HCoV-NL63, HCoV-HKU1, and HCoV-229E. The HCoV detection rates varied significantly with age (p = 0.00005), with the age group 0-14 years accounting for 28.6% (n = 30) of HCoV-positive patients. Fever and malaise were less frequent in HCoV patients than in influenza patients, while sore throat, dyspnoea, rhinorrhoea, and conjunctivitis were more associated with HCoV positivity. In conclusion, this study demonstrates that HCoV subtypes appear in ARI/ILI patients seen in general practice, with characteristic outbreak patterns primarily in winter. This study also identified symptoms associated with HCoVs in patients with ARI/ILI. Further studies with representative samples should be conducted to provide additional insights into the epidemiology and clinical features of HCoVs.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus 229E, Human/isolation & purification , Coronavirus Infections/epidemiology , Coronavirus NL63, Human/isolation & purification , Coronavirus OC43, Human/isolation & purification , Respiratory Tract Infections/epidemiology , Adolescent , Adult , Aged , Child , Child, Preschool , Coronavirus Infections/diagnosis , Disease Outbreaks , Female , Humans , Infant , Infant, Newborn , Influenza, Human/epidemiology , Male , Middle Aged , Nasopharynx/virology , Primary Health Care , Real-Time Polymerase Chain Reaction , Respiratory Tract Infections/diagnosis , Respiratory Tract Infections/virology , SARS-CoV-2 , Seasons , Young Adult
18.
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
19.
J Infect Dis ; 225(5): 768-776, 2022 03 02.
Article in English | MEDLINE | ID: covidwho-1722480

ABSTRACT

BACKGROUND: We determined the burden of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in air and on surfaces in rooms of patients hospitalized with coronavirus disease 2019 (COVID-19) and investigated patient characteristics associated with SARS-CoV-2 environmental contamination. METHODS: Nasopharyngeal swabs, surface, and air samples were collected from the rooms of 78 inpatients with COVID-19 at 6 acute care hospitals in Toronto from March to May 2020. Samples were tested for SARS-CoV-2 ribonucleic acid (RNA), cultured to determine potential infectivity, and whole viral genomes were sequenced. Association between patient factors and detection of SARS-CoV-2 RNA in surface samples were investigated. RESULTS: Severe acute respiratory syndrome coronavirus 2 RNA was detected from surfaces (125 of 474 samples; 42 of 78 patients) and air (3 of 146 samples; 3 of 45 patients); 17% (6 of 36) of surface samples from 3 patients yielded viable virus. Viral sequences from nasopharyngeal and surface samples clustered by patient. Multivariable analysis indicated hypoxia at admission, polymerase chain reaction-positive nasopharyngeal swab (cycle threshold of ≤30) on or after surface sampling date, higher Charlson comorbidity score, and shorter time from onset of illness to sampling date were significantly associated with detection of SARS-CoV-2 RNA in surface samples. CONCLUSIONS: The infrequent recovery of infectious SARS-CoV-2 virus from the environment suggests that the risk to healthcare workers from air and near-patient surfaces in acute care hospital wards is likely limited.


Subject(s)
COVID-19 , Nasopharynx/virology , SARS-CoV-2/isolation & purification , Adult , Aged , Air Microbiology , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19/transmission , COVID-19 Nucleic Acid Testing , Canada/epidemiology , Environmental Exposure , Health Personnel , Humans , Inpatients , Middle Aged , Pandemics/prevention & control , SARS-CoV-2/genetics
20.
Anal Chem ; 94(10): 4218-4226, 2022 03 15.
Article in English | MEDLINE | ID: covidwho-1721377

ABSTRACT

The most common diagnostic method used for coronavirus disease-2019 (COVID-19) is real-time reverse transcription polymerase chain reaction (PCR). However, it requires complex and labor-intensive procedures and involves excessive positive results derived from viral debris. We developed a method for the direct detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs, which uses matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-ToF MS) to identify specific peptides from the SARS-CoV-2 nucleocapsid phosphoprotein (NP). SARS-CoV-2 viral particles were separated from biological molecules in nasopharyngeal swabs by an ultrafiltration cartridge. Further purification was performed by an anion exchange resin, and purified NP was digested into peptides using trypsin. The peptides from SARS-CoV-2 that were inoculated into nasopharyngeal swabs were detected by MALDI-ToF MS, and the limit of detection was 106.7 viral copies. This value equates to 107.9 viral copies per swab and is approximately equivalent to the viral load of contagious patients. Seven NP-derived peptides were selected as the target molecules for the detection of SARS-CoV-2 in clinical specimens. The method detected between two and seven NP-derived peptides in 19 nasopharyngeal swab specimens from contagious COVID-19 patients. These peptides were not detected in four specimens in which SARS-CoV-2 RNA was not detected by PCR. Mutated NP-derived peptides were found in some specimens, and their patterns of amino acid replacement were estimated by accurate mass. Our results provide evidence that the developed MALDI-ToF MS-based method in a combination of straightforward purification steps and a rapid detection step directly detect SARS-CoV-2-specific peptides in nasopharyngeal swabs and can be a reliable high-throughput diagnostic method for COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Lasers , Nasopharynx , RNA, Viral/genetics , Specimen Handling/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
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