Project description:Novel Corona virus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or 2019-nCoV), and the subsequent disease caused by the virus (coronavirus disease 2019 or COVID-19), is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for SARS-CoV-2 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in 30-45 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. RNA isolated from nasopharyngeal swabs collected from actual COVID-19 patients was also tested. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected SARS-CoV-2 in both simulated patient samples and clinical specimens. This test was performed in 30-45 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.
Project description:Access to rapid and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is essential for controlling the current global pandemic of coronavirus disease 2019. In this study, the use of oral rinses (ORs) and posterior oropharyngeal saliva as an alternative to swab collection methods from symptomatic and asymptomatic health care workers for the detection of SARS-CoV-2 RNA by RT-PCR was evaluated. For saliva samples, the overall agreement with oropharyngeal swabs was 93% (? = 0.84), with a sensitivity of 96.7% (95% CI, 83.3%-99.8%). The agreement between saliva and nasopharyngeal swabs was 97.7% (? = 0.93), with a sensitivity of 94.1% (95% CI, 73.0%-99.7%). ORs were compared with nasopharyngeal swabs only, with an overall agreement of 85.7% (? = 0.65), and a sensitivity of 63% (95% CI, 46.6%-77.8%). The agreement between a laboratory-developed test based on the CDC RT-PCR and two commercial assays, the Xpert Xpress SARS-CoV-2 and the Cobas SARS-CoV-2, was also evaluated. The overall agreement was >90%. Finally, SARS-CoV-2 RNA in saliva samples was shown to be stable, with no changes in viral loads over 24 hours at both room temperature and 4°C. Although the dilution of SARS-CoV-2 in ORs precluded its acceptability as a sample type, posterior oropharyngeal saliva was an acceptable alternative sample type for SARS-CoV-2 RNA detection.
Project description:BACKGROUND:Amplification of viral ribonucleic acid (RNA) by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is the gold standard to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the initial outbreak, strategies to detect and isolate patients have been important to avoid uncontrolled viral spread. Although testing capacities have been upscaled, there is still a need for reliable high throughput test systems, specifically those that require alternative consumables. Therefore, we tested and compared two different methods for the detection of viral PCR products: rRT-PCR and mass spectrometry (MS). METHODS:Viral RNA was isolated and amplified from oro- or nasopharyngeal swabs. A total of 22 samples that tested positive and 22 samples that tested negative for SARS-CoV-2 by rRT-PCR were analyzed by MS. Results of the rRT-PCR and the MS protocol were compared. RESULTS:Results of rRT-PCR and the MS test system were in concordance in all samples. Time-to-results was faster for rRT-PCR. Hands-on-time was comparable in both assays. CONCLUSIONS:MS is a fast, reliable and cost-effective alternative for the detection of SARS-CoV-2 from oral and nasopharyngeal swabs.
Project description:OBJECTIVE:To determine whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is present in the vaginal secretions of both reproductive-aged and postmenopausal women during acute SARS-CoV-2 infection. DESIGN:Prospective study. SETTING:A single tertiary, university-affiliated medical centre in Israel. Time period, 1 June 2020 through to 31 July 2020. POPULATION:Women who were hospitalised in a single tertiary medical centre, who were diagnosed with acute SARS-CoV-2 infection by a nasopharyngeal RT-PCR test. METHODS:Women were diagnosed with acute SARS-CoV-2 infection by a nasopharyngeal RT-PCR test. Vaginal RT-PCR swabs were obtained from all study participants after a proper cleansing of the perineum. MAIN OUTCOME MEASURES:Detection of SARS-CoV-2 in vaginal RT-PCR swabs. RESULTS:Vaginal and nasopharyngeal swabs were obtained from 35 women, aged 21-93 years. Twenty-one women (60%) were in their reproductive years, of whom, five were in their third trimester of pregnancy. Most of the participants (57%) were healthy without any underlying medical conditions. Of the 35 patients sampled, 2 (5.7%) had a positive vaginal RT-PCR for SARS-CoV-2, one was premenopausal and the other was a postmenopausal woman. Both women had mild disease. CONCLUSION:Our findings contradict most previous reports, which did not detect the presence of viral colonisation in the vagina. Although passage through the birth canal exposes neonates to the vaginal polymicrobial flora, an acquisition of pathogens does not necessarily mandate neonatal infection or clinical disease. Nevertheless, when delivering the infant of a woman with acute SARS-CoV-2 infection, a clinician should consider the possibility of vaginal colonisation, even if it is uncommon. TWEETABLE ABSTRACT:When delivering the infant of a woman with acute SARS-CoV-2 infection, a clinician should consider the possibility of vaginal colonisation.
Project description:RATIONALE:Coronavirus disease 2019 (COVID-19), now a global pandemic, has spread to a large number of countries around the world. Symptoms of COVID-19 can range from mild to severe, including fever, cough, shortness of breath, and pneumonia. Some cases even remain asymptomatic. Data regarding the epidemiological and clinical features of children with COVID-19 are limited. Symptoms in children are thought to be atypical when compared with adults. As a result, diagnosis in many children is likely to be missed. Children presenting with atypical symptoms, especially those with a history of exposure, should be referred to early screening. PATIENT CONCERNS:A 23-month-old boy presented with a 2-day history of diarrhea. Chest computed tomography scan showed pneumonia. After admission to the hospital, the patient exhibited no diarrhea or other symptoms. Positive presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, was confirmed by 5 consecutive rounds of nucleic acid amplification testing of nasopharyngeal swabs. The patient was also found to have liver damage. DIAGNOSIS:Swabs were obtained for detection of SARS-CoV-2 RNA by established methods. INTERVENTIONS:Chinese traditional medicine prescription OUTCOMES:: Following treatment, signs of pneumonia on computed tomography scans were observed to be partially absorbed, and 2 consecutive rounds of nucleic acid amplification testing of swab samples were negative. The patient was discharged on the 21st day after admission to the hospital. On the 21st day after discharge, the patient had no recurrence of disease, no recurrence of pulmonary lesions, and normal liver function. CONCLUSION:This case study suggests that diarrhea not explained by common causes, such as acute gastroenteritis, could be a preliminary symptom of SARS-CoV-2 infection in children. Despite the lack of the presence of a fever or cough, lung pulmonary lesions were present in this child. SARS-CoV-2 infection may also cause hepatic injury. Even during the SARS-CoV-2 pneumonia recovery period, IgM and IgG antibodies can be positive for a long time.
Project description:We present the case of a 51-year-old patient with acute pericarditis as the dominant manifestation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The patient was admitted to the emergency department during a coronavirus disease 2019 (COVID-19) outbreak with a suspected ST-elevation myocardial infarction. A coronary angiogram was normal. Real-time reverse transcriptase PCR for the detection of nucleic acid from SARS-CoV-2 in a nasopharyngeal swab was positive. Laboratory tests revealed an increased white blood cell count, with neutrophilia and lymphocytopenia, elevated level of C-reactive protein, borderline elevated erythrocyte sedimentation rate, and slightly elevated interleukin 6. Echocardiography showed a hyperechogenic pericardium posterolaterally with minimal localized pericardial effusion. A chest computed tomography scan showed a small zone of ground-glass opacity in the right lower lobe (classified as CO-RADS 3). In patients with chest pain, ST elevation on electrocardiogram, a normal coronary angiogram, and suspected COVID-19, we should think of pericarditis as an unusual presentation of SARS-CoV-2 infection.
Project description:Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) and represents a global pandemic affecting more than 26 million people and has claimed >870,000 lives worldwide. Diagnostic tests for SARS-COV-2 infection commonly use nasopharyngeal swabs (NPS). As an alternative specimen, we investigated the potential use of the real-time reverse transcriptase PCR (RT-PCR) detection of SARS-COV-2 in saliva samples in large suspected-COVID-19 patients in Kuwait. NPS and saliva samples pairs were prospectively collected from 891 COVID-19 suspected patients in Kuwait and analyzed using TaqPath™ COVID-19 multiplex RT-PCR. Of the 891 patients, 38.61 % (344/891) were positive for SARS-CoV-2, 4.83 % (43/891) were equivocal, and 56.56 % (504/891) were negative with NPS by RT-PCR. For saliva, 34.23 % (305/891) were positive for SARS-CoV-2, 3.14 (28/891) were equivocal, and 62.63 % (558/891) were negative. From 344 confirmed cases for SARS-CoV-2 with NPS samples, 287 (83.43 %) (95 % CI, 79.14-86.99) were positive with saliva specimens. Moreover, the diagnostic sensitivity and specificity of RT-PCR for the diagnosis of COVID-19 in saliva were 83.43 % (95 % CI: 79.07-87.20) and 96.71 % (95 % CI: 94.85-98.04 %), respectively. An analysis of the agreement between the NPS and saliva specimens demonstrated 91.25 % observed agreement (? coefficient = 0.814, 95 % CI, 0.775-0.854). This study demonstrates that saliva can be a noninvasive specimen for detection of SARS-CoV-2 by RT-PCR.
Project description:Severe acute respiratory syndrome virus (SARS-CoV) that lacks the envelope (E) gene (rSARS-CoV-ΔE) is attenuated in vivo [1,2]. To identify factors that contribute to rSARS-CoV-ΔE attenuation, gene expression in cells infected by SARS-CoV with or without E gene was compared. Twenty-five stress response genes were preferentially upregulated during infection in the absence of the E gene. In addition, genes involved in signal transduction, transcription, cell metabolism, immunoregulation, inflammation, apoptosis and cell cycle and differentiation were differentially regulated in cells infected with rSARS-CoV with or without the E gene. Administration of E protein in trans reduced the stress response in cells infected with rSARS-CoV-ΔE, with respiratory syncytial virus, or treated with drugs, such as tunicamycin and thapsigarcin that elicit cell stress by different mechanisms. In addition, SARS-CoV E protein down-regulated the signaling pathway inositol-requiring enzyme 1 (IRE-1) of the unfolded protein response, but not the PKR-like ER kinase (PERK) or activating transcription factor 6 (ATF-6) pathways, and reduced cell apoptosis. Overall, the activation of the IRE-1 pathway was not able to restore cell homeostasis, and apoptosis was induced probably as a meassure to protect the host by limiting virus production and dissemination. The expression of proinflammatory cytokines was reduced in rSARS-CoV-ΔE-infected cells compared to rSARS-CoV-infected cells, suggesting that the increase in stress responses and the reduction of inflammation in the absence of the E gene contributed to the attenuation of rSARS-CoV-ΔE. We used Affymetrix microarrays (Human Genome U133 plus 2.0) to compare global gene expression between SARS-CoV-infected, mock-infected and SARS-CoV-ΔE-infected cells. For ech type of sample three hybridizations were carried-out (independent biological replicates).
Project description:Although the finding of severe acute respiratory syndrome coronavirus (SARS-CoV) in caged palm civets from live animal markets in China has provided evidence for interspecies transmission in the genesis of the SARS epidemic, subsequent studies suggested that the civet may have served only as an amplification host for SARS-CoV. In a surveillance study for CoV in noncaged animals from the wild areas of the Hong Kong Special Administration Region, we identified a CoV closely related to SARS-CoV (bat-SARS-CoV) from 23 (39%) of 59 anal swabs of wild Chinese horseshoe bats (Rhinolophus sinicus) by using RT-PCR. Sequencing and analysis of three bat-SARS-CoV genomes from samples collected at different dates showed that bat-SARS-CoV is closely related to SARS-CoV from humans and civets. Phylogenetic analysis showed that bat-SARS-CoV formed a distinct cluster with SARS-CoV as group 2b CoV, distantly related to known group 2 CoV. Most differences between the bat-SARS-CoV and SARS-CoV genomes were observed in the spike genes, ORF 3 and ORF 8, which are the regions where most variations also were observed between human and civet SARS-CoV genomes. In addition, the presence of a 29-bp insertion in ORF 8 of bat-SARS-CoV genome, not in most human SARS-CoV genomes, suggests that it has a common ancestor with civet SARS-CoV. Antibody against recombinant bat-SARS-CoV nucleocapsid protein was detected in 84% of Chinese horseshoe bats by using an enzyme immunoassay. Neutralizing antibody to human SARS-CoV also was detected in bats with lower viral loads. Precautions should be exercised in the handling of these animals.
Project description:The aim of this study was to compare the sensitivity of self-collected versus healthcare worker (HCW)-collected swabs for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) testing. Symptomatic individuals referred for SARS-CoV-2 testing were invited to provide mobile-phone video-instructed self-collected oropharyngeal and nasal samples followed by a HCW-collected oropharyngeal sample. All samples were sent for analysis to the same microbiology laboratory, and the number of SARS-CoV-2-positive participants in the two tests was compared. A total of 109 participants were included, and 19 participants had SARS-CoV-2-positive results. The diagnostic sensitivity of the self-collected and HCW-collected swabs was 84.2% and 89.5%, respectively, with an acceptable agreement, Cohens kappa 0.82, p < 0.001. Further, results from a questionnaire answered by the participants found that loss of smell as a self-reported symptom was a strong predictor for a SARS-CoV-2-positive test. In conclusion, we found that self-collected oropharyngeal and nasal swabs for SARS-CoV-2 testing can be reliable compared to HCW-collected oropharyngeal samples.