Nasopharyngeal Swabs Of Patients Research Articles

SARS-CoV-2 produces a microRNA CoV2-miR-O8 in patients with COVID-19 infection

Many viruses produce microRNAs (miRNAs), termed viral miRNAs (v-miRNAs), with the capacity to target host gene expression. Bioinformatic and cell culture studies suggest that SARS-CoV-2 can also generate v-miRNAs. This patient-based study defines the SARS-CoV-2 encoded small RNAs present in nasopharyngeal swabs of patients with COVID-19 infection using small RNA-seq. A specific conserved sequence (CoV2-miR-O8) is defined that is not expressed in other coronaviruses but is preserved in all SARS-CoV-2 variants. CoV2-miR-O8 is highly represented in nasopharyngeal samples from patients with COVID-19 infection, is detected by RT-PCR assays in patients, has features consistent with Dicer and Drosha generation as well as interaction with Argonaute and targets specific human microRNAs.

Near complete genome sequences from Southern Vietnam revealed local features of genetic diversity and intergenerational changes in SARS- CoV-2 variants in 2020–2021

BackgroundSince its beginnings in 2019, the COVID-19 pandemic is still a problem of global medical concern. Southern Vietnam is one of the country's vast regions, including 20 provinces and the densely populated metropolis Ho Chi Minh City. A randomized retrospective study was performed to investigate the epidemiology and genetic diversity of COVID-19. Whole-genome sequencing of 126 SARS-CoV-2 samples collected from Southern Vietnam between January 2020 and December 2021 revealed the main circulating variants and their distribution.MethodsEpidemiological data were obtained from the Department of Preventive Medicine of the Vietnamese Ministry of Health. To identify circulating variants, RNA, extracted from 126 nasopharyngeal swabs of patients with suspected COVID-19 were sequenced on Illunina MiSeq to obtain near complete genomes SARS-CoV-2.ResultsDue to the effectiveness of restrictive measures in Vietnam, it was possible to keep incidence at a low level. The partial relaxation of restrictive measures, and the spread of Delta lineages, contributed to the beginning of a logarithmic increase in incidence. Lineages 20A-H circulated in Southern Vietnam during 2020. Spread of the Delta lineage in Southern Vietnam began in March 2021, causing a logarithmic rise in the number of COVID-19 cases.ConclusionsPandemic dynamics in Southern Vietnam feature specific variations in incidence, and these reflect the success of the restrictive measures put in place during the early stages of the pandemic.

TRIM29 acts as a potential senescence suppressor with epigenetic activation in nasopharyngeal carcinoma.

Epigenetic alterations marked by DNA methylation are frequent events during the early development of nasopharyngeal carcinoma (NPC). We identified that TRIM29 is hypomethylated and overexpressed in NPC cell lines and tissues. TRIM29 silencing not only limited the growth of NPC cells in vitro and in vivo, but also induced cellular senescence, along with reactive oxygen species (ROS) accumulation. Mechanistically, we found that TRIM29 interacted with voltage-dependent anion-selective channel 1 (VDAC1) to activate mitophagy clearing up damaged mitochondria, which are the major source of ROS. In patients with NPC, high levels of TRIM29 expression are associated with an advanced clinical stage. Moreover, we detected hypomethylation of TRIM29 in patient nasopharyngeal swab DNA. Our findings indicate that TRIM29 depends on VDAC1 to induce mitophagy and prevents cellular senescence by decreasing ROS. Detection of aberrantly methylated TRIM29 in the nasopharyngeal swab DNA could be a promising strategy for the early detection of NPC.

Genes Involved in miRNA Biogenesis Are Not Downregulated in SARS-CoV-2 Infection.

miRNAs, small non-coding RNAs that regulate gene expression, are involved in various pathological processes, including viral infections. Virus infections may interfere with the miRNA pathway through the inhibition of genes involved in miRNA biogenesis. A reduction in the number and the levels of miRNAs expressed in nasopharyngeal swabs of patients with severe COVID-19 was lately observed by us, pointing towards the potential of miRNAs as possible diagnostic or prognostic biomarkers for predicting outcomes among patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The objective of the present study was to investigate whether SARS-CoV-2 infection influences the expression levels of messenger RNAs (mRNAs) of key genes involved in miRNA biogenesis. mRNA levels of AGO2, DICER1, DGCR8, DROSHA, and Exportin-5 (XPO5) were measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) in nasopharyngeal swab specimens from patients with COVID-19 and controls, as well as in cells infected with SARS-CoV-2 in vitro. Our data showed that the mRNA expression levels of AGO2, DICER1, DGCR8, DROSHA, and XPO5 were not significantly different in patients with severe COVID-19 when compared to patients with non-severe COVID-19 and controls. Similarly, the mRNA expression of these genes was not affected by SARS-CoV-2 infection in NHBE and Calu-3 cells. However, in Vero E6 cells, AGO2, DICER1, DGCR8, and XPO5 mRNA levels were slightly upregulated 24 h after infection with SARS-CoV-2. In conclusion, we did not find evidence for downregulation of mRNA levels of miRNA biogenesis genes during SARS-CoV-2 infection, neither ex vivo nor in vitro.

Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation.

Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/μL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing.

Dual-Domain Reporter Approach for Multiplex Identification of Major SARS-CoV-2 Variants of Concern in a Microarray-Based Assay

Since the emergence of the COVID-19 pandemic in December 2019, the SARS-CoV-2 virus continues to evolve into many variants emerging around the world. To enable regular surveillance and timely adjustments in public health interventions, it is of the utmost importance to accurately monitor and track the distribution of variants as rapidly as possible. Genome sequencing is the gold standard for monitoring the evolution of the virus, but it is not cost-effective, rapid and easily accessible. We have developed a microarray-based assay that can distinguish known viral variants present in clinical samples by simultaneously detecting mutations in the Spike protein gene. In this method, the viral nucleic acid, extracted from nasopharyngeal swabs, after RT-PCR, hybridizes in solution with specific dual-domain oligonucleotide reporters. The domains complementary to the Spike protein gene sequence encompassing the mutation form hybrids in solution that are directed by the second domain ("barcode" domain) at specific locations on coated silicon chips. The method utilizes characteristic fluorescence signatures to unequivocally differentiate, in a single assay, different known SARS-CoV-2 variants. In the nasopharyngeal swabs of patients, this multiplex system was able to genotype the variants which have caused waves of infections worldwide, reported by the WHO as being of concern (VOCs), namely Alpha, Beta, Gamma, Delta and Omicron variants.

1914. Characteristics of Hospital Onset SARS Cov-2 Infections Before and After the Emergence of The Highly Transmissible Variant B.1.1.529 In a Comprehensive Cancer Center

Abstract Background SARS-CoV-2 B.1.1.529 (Omicron) variant was first identified in November 2021 in South Africa and was notable for its increased transmissibility and rapid spread worldwide. In the United States, this variant led to a surge in COVID-19 cases by December 2021. As a result, we experienced a steep rise in cases among patients and employees at our institution starting December 22nd, 2021. Therefore, we compared the incidence and characteristics of hospital-onset COVID-19 (HO-COVID-19) in our cancer patients prior to and during the surge of the Omicron variant. Methods We identified HO-COVID-19, as per the CDC definition, from our infection control surveillance database, and additional contact tracing information was reviewed to determine the possible sources of HO-COVID-19. Whole-genome sequencing studies were conducted randomly on nasopharyngeal swabs of patients and employees who had COVID-19 during the study period. Results Twenty-six HO-COVID-19 infections were identified from the beginning of the pandemic (February 2020) through February 2022 (Table 1). Only 17 cases occurred over 22 months from the beginning of the pandemic through early December 2021 (Figure 1). These HO-COVID-19 occurred during the 3 COVID-19 surges that were epidemiologically attributed to the variants seen prior to Omicron. Among these 17 patients, 12 (70%) were symptomatic, 9 (53%) had a link to an infected employee, 7 (41%) died during their hospitalization (3 of the deaths were attributable to COVID-19), and 10 (59%) recovered and were discharged. Over 6 weeks (from December 22nd, 2021, through February 1st, 2022), 9 HO-COVID-19 were discovered during the Omicron variant surge (Figure 1). Six (67%) of these patients were symptomatic, 8 (89%) had a link to an infected employee, 2 (22%) died (1 death was attributed to COVID-19 ), and 7 (78%) recovered and were discharged. Table 1.Characteristics and demographics of patients with hospital-onset COVID-19.Abbreviation: PCR, polymerase chain reaction; HOCI, hospital-onset COVID-19 infections.Figure 1.Bar chart of nosocomial COVID-19 cases graphed against line graphs of COVID-19 infections diagnosed weekly among patient and employee at MD Anderson Cancer Center between March 2020 through February 2022 Conclusion The Omicron variant surge led to marked increases in HO-COVID-19 despite the continuous adoption of enhanced infection control practices, testing on admission, and daily symptoms screening of patients and employees. Disclosures Roy F. Chemaly, MD/MPH, Karius: Advisor/Consultant|Karius: Grant/Research Support.

A case\u2013control study of the causes of acute respiratory infection among hospitalized patients in Northeastern Laos

With the advent of highly sensitive real-time PCR, multiple pathogens have been identified from nasopharyngeal swabs of patients with acute respiratory infections (ARIs). However, the detection of microorganisms in the upper respiratory tract does not necessarily indicate disease causation. We conducted a matched case–control study, nested within a broader fever aetiology project, to facilitate determination of the aetiology of ARIs in hospitalised patients in Northeastern Laos. Consenting febrile patients of any age admitted to Xiengkhuang Provincial Hospital were included if they met the inclusion criteria for ARI presentation (at least one of the following: cough, rhinorrhoea, nasal congestion, sore throat, difficulty breathing, and/or abnormal chest auscultation). One healthy control for each patient, matched by sex, age, and village of residence, was recruited for the study. Nasopharyngeal swabs were collected from participants and tested for 33 pathogens by probe-based multiplex real-time RT-PCR (FastTrack Diagnostics Respiratory pathogen 33 kit). Attributable fraction of illness for a given microorganism was calculated by comparing results between patients and controls (= 100 * [OR − 1]/OR) (OR = odds ratio). Between 24th June 2019 and 24th June 2020, 205 consenting ARI patients and 205 matching controls were recruited. After excluding eight pairs due to age mismatch, 197 pairs were included in the analysis. Males were predominant with sex ratio 1.2:1 and children < 5 years old accounted for 59% of participants. At least one potential pathogen was detected in 173 (88%) patients and 175 (89%) controls. ARI in admitted patients were attributed to influenza B virus, influenza A virus, human metapneumovirus (HMPV), and respiratory syncytial virus (RSV) in 17.8%, 17.2%, 7.5%, and 6.5% of participants, respectively. SARS-CoV-2 was not detected in any cases or controls. Determining ARI aetiology in individual patients remains challenging. Among hospitalised patients with ARI symptoms presenting to a provincial hospital in Northeastern Laos, half were determined to be caused by one of several respiratory viruses, in particular influenza A virus, influenza B virus, HMPV, and RSV.

Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs.

The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity.

A benchmarking study of SARS-CoV-2 whole-genome sequencing protocols using COVID-19 patient samples

SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging new type of coronavirus that is responsible for the COVID-19 pandemic and the unprecedented global health emergency. Whole-genome sequencing (WGS) of SARS-CoV-2 plays a critical role in understanding the disease. Performance variation exists across SARS-CoV-2 viral WGS technologies, but there is currently no benchmarking study comparing different WGS sequencing protocols. We compared seven different SARS-CoV-2 WGS library protocols using RNA from patient nasopharyngeal swab samples under two storage conditions with low and high viral inputs. We found large differences in mappability and genome coverage, and variations in sensitivity, reproducibility, and precision of single-nucleotide variant calling across different protocols. For certain amplicon-based protocols, an appropriate primer trimming step is critical for accurate single-nucleotide variant calling. We ranked the performance of protocols based on six different metrics. Our findings offer guidance in choosing appropriate WGS protocols to characterize SARS-CoV-2 and its evolution.

A 17-Year-Old Girl with a Recent History of Marijuana Use Presented with Pneumomediastinum and Pneumopericardium and Tested Positive for SARS-CoV-2 Infection on Hospital Admission

Patient: Female, 17-year-oldFinal Diagnosis: Pneumomediastinum • pneumopericardiumSymptoms: Abdominal pain • diarrhea • nausea • vomitingMedication: —Clinical Procedure: Recovered • symptomatic and supportive careSpecialty: Pediatrics and NeonatologyObjective:Unusual clinical courseBackground:Pneumomediastinum and pneumopericardium have been reported to occur in people who regularly smoke marijuana and have also been reported in patients with COVID-19 pneumonia due to infection with SARSCoV-2. This report is of a 17-year-old girl with a history of marijuana use who presented with pneumomediastinum and pneumopericardium and was found to be positive for SARS-CoV-2 infection on hospital admission by Abbott ID NOW testing.Case Report:A 17-year-old girl presented to the emergency room with a 3-day history of abdominal pain, nausea, and vomiting and a 1-day history of diarrhea. She had a history of daily marijuana use and lived with her grandmother who was presumed to be positive for COVID-19, based on symptoms. Her admission laboratory results were unremarkable except for pyuria, which was suspicious for urinary tract infection. The patient’s nasopharyngeal swab was positive for SARS-CoV-2 infection. Owing to abdominal pain, a computed tomography (CT) scan of the abdomen and pelvis was obtained, which was concerning for pneumomediastinum and pneumopericardium. A CT scan of the thorax confirmed the findings. A contrast-enhanced barium esophagogram was performed and was unremarkable. The patient was admitted to the pediatric intensive care unit for observation and supportive care.Conclusions:This report shows the importance of current testing for SARS-CoV-2 infection in patients of all ages who present acutely to the hospital. It also highlights the importance of obtaining a full social and medical history so that symptoms and signs from causes other than SARS-CoV-2 infection are not missed.

АЛГОРИТМ ВИДОВОЙ ДИФФЕРЕНЦИАЦИИ ВОЗБУДИТЕЛЕЙ РОДА BORDETELLA И РЕЗУЛЬТАТЫ ЕГО ИСПОЛЬЗОВАНИЯ В РЕСПУБЛИКЕ БЕЛАРУСЬ

Species-specific differentiation of Bordetella pathogens in nasopharyngeal swabs of patients with suspected whooping cough was carried out using developed by us real-time PCR: duplex TaqMan PCR aimed for detecting B. pertussis and B. parapertussis, with primers targeting IS481, IS1001, BP0026 and GAPDH as the human internal control and two singleplex TaqMan PCR aimed for detecting B. holmesii and B. bronchiseptica with primers targeting hIS1001 and flaA specific genes. Among the 3130 throat swab analyzed, B. pertussis, B. parapertussis and B. holmesii were detected in 29.5 %, 0.9 % and 0.4 % of samples respectively. The developed algorithm proved to be a useful molecular-genetic diagnostic tool for whooping cough, established cocirculation of different species of Bordetella genus and demonstrated the predominance of B. pertussis, low rate of B. parapertussis and B. holmesii and the absence of B. bronchiseptica in the circulation in the Republic of Belarus.

Negative SARS-CoV2-antibodies after positive COVID-19-PCR nasopharyngeal swab in patients treated with anti-CD20 therapies.

Negative SARS-CoV2-antibodies after positive COVID-19-PCR nasopharyngeal swab in patients treated with anti-CD20 therapies.

Detection of SARS-CoV-2 RNA in nasopharyngeal swabs from COVID-19 patients and asymptomatic cases of infection by real-time and digital PCR.

In this work we tested two reagent kits developed by us for detecting SARS-CoV-2 RNA using a fragment of the ORF1ab gene in digital PCR and real-time PCR formats. Data were obtained on the detection of SARS-CoV-2 virus RNA in nasopharyngeal swabs of patients with COVID-19 and asymptomatic carriers. The developed reagent kits provided 100% sensitivity and a detection limit of 103 GE / ml for qPCR, and at least 200 copies / ml of viral RNA when performing digital PCR. These methods were tested using a panel of 1,328 samples collected from patients with suspected COVID-19 at the beginning of 2020 in the Russian Federation. It has been shown that dPCR is more sensitive and can be used to analyze samples with low viral load, including those from patients without clinical symptoms. dPCR significantly improves the accuracy of laboratory research and significantly reduces the number of false negative results in the diagnosis of SARS-CoV-2. Determination of the concentration of SARS-CoV-2 RNA in patients with different clinical course of the disease showed that the concentration of viral RNA can sharply decrease in the first days of the disease. A low concentration of viral RNA in samples from patients is also characteristic of asymptomatic disease. Digital PCR provides a higher detection rate for asymptomatic cases, which is approximately 75% of those infected, as opposed to 45% for real-time PCR. The results obtained on the use of the digital PCR method for detecting SARS-CoV-2 RNA showed that this method is especially suitable for detecting RNA in case of its low concentration in contacts, as well as for monitoring changes in viral load in convalescent patients.

Host transcriptomic profiling of COVID-19 patients with mild, moderate, and severe clinical outcomes

Characterizing key molecular and cellular pathways involved in COVID-19 is essential for disease prognosis and management. We perform shotgun transcriptome sequencing of human RNA obtained from nasopharyngeal swabs of patients with COVID-19, and identify a molecular signature associated with disease severity. Specifically, we identify globally dysregulated immune related pathways, such as cytokine-cytokine receptor signaling, complement and coagulation cascades, JAK-STAT, and TGF- β signaling pathways in all, though to a higher extent in patients with severe symptoms. The excessive release of cytokines and chemokines such as CCL2, CCL22, CXCL9 and CXCL12 and certain interferons and interleukins related genes like IFIH1, IFI44, IFIT1 and IL10 were significantly higher in patients with severe clinical presentation compared to mild and moderate presentations. Differential gene expression analysis identified a small set of regulatory genes that might act as strong predictors of patient outcome. Our data suggest that rapid transcriptome analysis of nasopharyngeal swabs can be a powerful approach to quantify host molecular response and may provide valuable insights into COVID-19 pathophysiology.

The Role of Flexible Bronchoscopy in Swab-negative Patients During the SARS-CoV2 Pandemic.

The Role of Flexible Bronchoscopy in Swab-negative Patients During the SARS-CoV2 Pandemic.

COVID-19 and risk of arrhythmia?

To the Editor Coronavirus disease-2019 (COVID-19) is an infectious disease caused by severe acute respiratory coronavirus 2 (SARS-CoV-2) with a wide range of clinical manifestations, mainly dominated by respiratory symptoms.1 With the growing number of infected patients, major cardiac complications have been reported2,3 with higher risk of in-hospital mortality.4 An 85-year-old male with a history of hypertension, type 2 diabetes and junctional rhythm (Fig. 1A) presented to the ED due to progressive dyspnea and dry cough in the previous week and paroxysmal nocturnal dyspnea on the previous day. The physical examination revealed a blood pressure of 118/45 mm Hg, heart rate of 36 bpm, O2 saturation of 90% on room air and an axillary temperature of 37.7°C. The patient had bilateral crackles on lung auscultation. Arterial blood gas analysis showed type 1 respiratory failure. A 12-lead electrocardiogram (EKG) showed junctional rhythm with left bundle branch block (Fig. 1B). Blood-work revealed an elevated C-reactive protein (83.8 mg/dL; normal ≤7.5 mg/L), D-dimer (2095 ng/mL; normal <243 ng/mL), N-terminal pro-brain natriuretic peptide (5360 pg/mL; normal ≤133 pg/mL), creatinine phosphokinase (760 UI/L; normal 10–72 UI/L) and high sensitivity troponin (139 pg/mL; normal <19.8 pg/mL). The chest x-ray showed an enlarged cardiothoracic ratio. The patient's nasopharyngeal swab was positive for SARS-CoV-2 PCR. The patient had a favorable chronotropic response to inhaled salbutamol and was admitted to the COVID ward under cardiac telemetry monitoring. On the first day of admission, a new episode of bradycardia was observed. The EKG showed a complete left bundle branch block and a permanent pacemaker was placed. The remainder of his hospital stay was uneventful with resolution of heart failure symptoms, respiratory failure and progressive decrease of the inflammatory markers and troponin levels.Figure 1: A, Previous 12-lead electrocardiogram (2015)—junctional rhythm. B, 12-Lead electrocardiogram on admission (May 2020)—junctional rhythm, enlargement of QRS complex on D1, aVL, V5 and V6 leads.Although the pathophysiology underlying COVID-19 remains poorly understood, SARS-CoV-2 may directly injure the heart leading to higher risk of in-hospital mortality.4 Some theories have been postulated to explain the myocardial injury and the damage to the conduction system.2 One explanation suggests that patients with chronic cardiovascular diseases may become unstable due to the imbalance between metabolic demand and reduced cardiac reserve, intensified by the inflammatory response and disturbance of autonomic tone.2 This injurious effect could be perpetuated by the prompt and severe downregulation of myocardial and pulmonary ACE2 pathways.2 Another matter of debate has been the use of drugs that have been tested in patients with COVID-19 that predispose to arrythmias, such as hydroxychloroquine and azithromycin.5 This was not the case since this patient was treated with supportive care alone without any antiviral or immunomodulatory therapies. In conclusion, we present the case of an elderly man with a predisposition to arrhythmia in whom SARS-CoV-2 infection was diagnosed when he presented to the ED with severe bradycardia. Could the conduction abnormality described in this patient (who already had a predisposition to arrhythmia) be interpreted as a result of the disfunction of the electrical conduction system induced by SARS-CoV-2? Or could it merely reflect the impact of a systemic illness like many others?6 Our knowledge regarding the cardiovascular involvement of SARS-CoV-2 infection is still limited. In these patients, one should remain on high alert for cardiovascular complications. Acknowledgments We acknowledge Mari Mesquita MD MSc and Mariana Meireles MD MSc for their helpful comments regarding this patient's case. Conflicts of interest MMB, DP, SG, BS, HV and LN declares no conflicts of interest, real or perceived, financial or nonfinancial

SARS-CoV-2 Whole Genome Amplification and Sequencing for Effective Population-Based Surveillance and Control of Viral Transmission.

BackgroundWith the gradual reopening of economies and resumption of social life, robust surveillance mechanisms should be implemented to control the ongoing COVID-19 pandemic. Unlike RT-qPCR, SARS-CoV-2 whole genome sequencing (cWGS) has the added advantage of identifying cryptic origins of the virus, and the extent of community-based transmissions versus new viral introductions, which can in turn influence public health policy decisions. However, the practical and cost considerations of cWGS should be addressed before it is widely implemented.MethodsWe performed shotgun transcriptome sequencing using RNA extracted from nasopharyngeal swabs of patients with COVID-19, and compared it to targeted SARS-CoV-2 genome amplification and sequencing with respect to virus detection, scalability, and cost-effectiveness. To track virus origin, we used open-source multiple sequence alignment and phylogenetic tools to compare the assembled SARS-CoV-2 genomes to publicly available sequences.ResultsWe found considerable improvement in whole genome sequencing data quality and viral detection using amplicon-based target enrichment of SARS-CoV-2. With enrichment, more than 99% of the sequencing reads mapped to the viral genome compared to an average of 0.63% without enrichment. Consequently, an increase in genome coverage was obtained using substantially less sequencing data, enabling higher scalability and sizable cost reductions. We also demonstrated how SARS-CoV-2 genome sequences can be used to determine their possible origin through phylogenetic analysis including other viral strains.ConclusionsSARS-CoV-2 whole genome sequencing is a practical, cost-effective, and powerful approach for population-based surveillance and control of viral transmission in the next phase of the COVID-19 pandemic.

Tracheal colonization factor A (TcfA) is a biomarker for rapid and specific detection of Bordetella pertussis

Pertussis is a highly contagious disease for which prompt, point-of-care diagnosis remains an unmet clinical need. Results from conventional test modalities (nucleic acid detection, serology, and culture) take hours to days. To overcome this challenge, we identified a new biomarker (tracheal colonization factor A, TcfA) for detection of Bordetella pertussis infection by lateral flow immunoassay (LFIA). We developed a library of 28 epitope-mapped monoclonal antibodies against TcfA and incorporated three antibodies into a LFIA. The LFIA did not cross-react with common bacterial or fungal organisms, but did react with nine distinct B. pertussis strains. The minimal linear epitope sequences targeted by the LFIA were conserved in 98% of 954 B. pertussis isolates collected across 12 countries from 1949–2017. The LFIA’s limit of detection was 3.0 × 105 CFU/mL with B. pertussis cells in buffer, 6.2 × 105 CFU/mL with nasopharyngeal washes from a non-human primate model, and 2.3 ng/mL with recombinant TcfA. The LFIA reacted with patient nasopharyngeal swab specimens containing as few as 1.8 × 106B. pertussis genomes/mL and showed no false-positives. Rapid (< 20 min) LFIA detection of TcfA as a biomarker for B. pertussis infection is feasible and may facilitate early detection of pertussis.

Viral Load Dynamics in Sputum and Nasopharyngeal Swab in Patients with COVID-19

Coronavirus disease 2019 (COVID-19) has caused a global pandemic associated with substantial morbidity and mortality. Nasopharyngeal swabs and sputum samples are generally collected for serial viral load screening of respiratory contagions, but temporal profiles of these samples are not completely clear in patients with COVID-19. We performed an observational cohort study at Renmin Hospital of Wuhan University, which involved 31 patients with confirmed COVID-19 with or without underlying diseases. We obtained samples from each patient, and serial viral load was measured by real-time quantitative polymerase chain reaction. We found that the viral load in the sputum was inclined to be higher than samples obtained from the nasopharyngeal swab at disease presentation. Moreover, the viral load in the sputum decreased more slowly over time than in the nasopharyngeal group as the disease progressed. Interestingly, even when samples in the nasopharyngeal swab turned negative, it was commonly observed that patients with underlying diseases, especially hypertension and diabetes, remained positive for COVID-19 and required a longer period for the sputum samples to turn negative. These combined findings emphasize the importance of tracking sputum samples even in patients with negative tests from nasopharyngeal swabs, especially for those with underlying conditions. In conclusion, this work reinforces the importance of sputum samples for SARS-CoV-2 detection to minimize transmission of COVID-19 within the community.