The characteristics of microbiome in the upper respiratory tract of COVID-19 patients.

BACKGROUND: Co-infection with other pathogens in coronavirus disease 2019 (COVID-19) patients exacerbates disease severity and impacts patient prognosis. Clarifying the exact pathogens co-infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is premise of the precise treatment for COVID-19 patients. METHODS: Sputum samples were collected from 17 patients in the COVID-19 positive group and 18 patients in the COVID-19 negative group. DNA extraction was performed to obtain the total DNA. Sequencing analysis using 16S and ITS rRNA gene was carried out to analyze the composition of bacterial and fungal communities. Meanwhile, all the samples were inoculated for culture. RESULTS: We did not observe significant differences in bacterial composition between the COVID-19 positive and negative groups. However, a significantly higher abundance of Candida albicans was observed in the upper respiratory tract samples from the COVID-19 positive group compared to the COVID-19 negative group. Moreover, the Candida albicans strains isolated from COVID-19 positive group exhibited impaired secretion of aspartyl proteinases. CONCLUSION: COVID-19 positive patients demonstrate a notable increase in the abundance of Candida albicans, along with a decrease in the levels of aspartyl proteinases, indicating the alteration of microbiota composition of upper respiratory tract.

Clinical findings, viral load, and outcomes of COVID-19: Comparison of patients with negative and positive initial chest computed tomography.

Reports detailing the clinical characteristics, viral load, and outcomes of patients with normal initial chest CT findings are lacking. We sought to compare the differences in clinical findings, viral loads, and outcomes between patients with confirmed COVID-19 who initially tested negative on chest CT (CT negative) with patients who tested initially positive on chest CT (CT positive). The clinical data, viral loads, and outcomes of initial CT-positive and CT-negative patients examined between January 2020 and April 2020 were retrospectively compared. The efficacy of viral load (cyclic threshold value [Ct value]) in predicting pneumonia was evaluated using receiver operating characteristic (ROC) curve and area under the curve (AUC). In total, 128 patients underwent initial chest CT (mean age, 54.3 ± 19.0 years, 50% male). Of those, 36 were initially CT negative, and 92 were CT positive. The CT-positive patients were significantly older (P < .001) than the CT-negative patients. Only age was significantly associated with the initial presence of pneumonia (odds ratio, 1.060; confidence interval (CI), 1.020-1-102; P = .003). In addition, age (OR, 1.062; CI, 1.014-1.112; P = .011), fever at diagnosis (OR, 6.689; CI, 1.715-26.096; P = .006), and CRP level (OR, 1.393; CI, 1.150-1.687; P = .001) were significantly associated with the need for O2 therapy. Viral load was significantly higher in the CT-positive group than in the CT-negative group (P = .017). The cutoff Ct value for predicting the presence of pneumonia was 27.71. Outcomes including the mean hospital stay, intensive care unit admission, and O2 therapy were significantly worse in the CT-positive group than in the CT-negative group (all P < .05). In conclusion, initially CT-negative patients showed better outcomes than initially CT-positive patients. Age was significantly associated with the initial presence of pneumonia, and viral load may help in predicting the initial presence of pneumonia.

Accuracy of Real-Time Polymerase Chain Reaction in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is a mild to severe respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The diagnostic accuracy of the Centers for Disease Control and Prevention (CDC)- or World Health Organization (WHO)-recommended real-time PCR (RT-qPCR) primers in clinical practice remains unproven. We conducted a prospective study on the accuracy of RT-qPCR using an in-house-designed primer set (iNP) targeting the nucleocapsid protein as well as various recommended and commercial primers. The accuracy was assessed by culturing or seroconversion. We enrolled 12 confirmed COVID-19 patients with a total of 590 clinical samples. When a cutoff value of the cycle threshold (Ct) was set to 35, RT-qPCRs with WHO RdRp primers and CDC N1, N2, and N3 primers showed sensitivity of 42.1% to 63.2% and specificity of 90.5% to 100% in sputum, and sensitivity of 65.2% to 69.6% and specificity of 65.2% to 69.6% in nasopharyngeal samples. The sensitivity and specificity of iNP RT-qPCR in sputum and nasopharyngeal samples were 94.8%/100% and 69.6%/100%, respectively. Sputum testing had the highest sensitivity, followed by nasopharyngeal testing (P = 0.0193); self-collected saliva samples yielded better characteristics than oropharyngeal samples (P = 0.0032). Our results suggest that iNP RT-qPCR has better sensitivity and specificity than RT-PCR with WHO (P < 0.0001) or CDC (N1: P = 0.0012, N2: P = 0.0013, N3: P = 0.0012) primers. Sputum RT-qPCR analysis has the highest sensitivity, followed by nasopharyngeal, saliva, and oropharyngeal assays. Our study suggests that considerable improvement is needed for the RT-qPCR WHO and CDC primer sets for detecting SARS-CoV-2. IMPORTANCE Numerous research campaigns have addressed the vast majority of clinical and diagnostic specificity and sensitivity of various primer sets of SARS-CoV2 viral detection. Despite the impressive progress made to resolve the pandemic, there is still a need for continuous and active improvement of primers used for diagnosis in clinical practice. Our study significantly exceeds the scale of previously published research on the specificity and sensitivity of different primers comparing with different specimens and is the most comprehensive to date in terms of constant monitoring of primer sets of current usage. Henceforth, our results suggest that sputum samples sensitivity is the highest, followed by nasopharyngeal, saliva, and oropharyngeal samples. The CDC recommends the use of oropharyngeal specimens, leading to certain discrepancy between the guidelines set forth by the CDC and IDSA. We proved that the oropharyngeal samples demonstrated the lowest sensitivity for the detection of SARS-CoV-2.

Development and Evaluation of AccuPower COVID-19 Multiplex Real-Time RT-PCR Kit and AccuPower SARS-CoV-2 Multiplex Real-Time RT-PCR Kit for SARS-CoV-2 Detection in Sputum, NPS/OPS, Saliva and Pooled Samples.

Rapid and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the successful control of the current global COVID-19 pandemic. The real-time reverse transcription polymerase chain reaction (Real-time RT-PCR) is the most widely used detection technique. This research describes the development of two novel multiplex real-time RT-PCR kits, AccuPower® COVID-19 Multiplex Real-Time RT-PCR Kit (NCVM) specifically designed for use with the ExiStation™48 system (comprised of ExiPrep™48 Dx and Exicycler™96 by BIONEER, Korea) for sample RNA extraction and PCR detection, and AccuPower® SARS-CoV-2 Multiplex Real-Time RT-PCR Kit (SCVM) designed to be compatible with manufacturers' on-market PCR instruments. The limit of detection (LoD) of NCVM was 120 copies/mL and the LoD of the SCVM was 2 copies/µL for both the Pan-sarbecovirus gene and the SARS-CoV-2 gene. The AccuPower® kits demonstrated high precision with no cross reactivity to other respiratory-related microorganisms. The clinical performance of AccuPower® kits was evaluated using the following clinical samples: sputum and nasopharyngeal/oropharyngeal swab (NPS/OPS) samples. Overall agreement of the AccuPower® kits with a Food and Drug Administration (FDA) approved emergency use authorized commercial kit (STANDARD™ M nCoV Real-Time Detection kit, SD BIOSENSOR, Korea) was above 95% (Cohen's kappa coefficient ≥ 0.95), with a sensitivity of over 95%. The NPS/OPS specimen pooling experiment was conducted to verify the usability of AccuPower® kits on pooled samples and the results showed greater than 90% agreement with individual NPS/OPS samples. The clinical performance of AccuPower® kits with saliva samples was also compared with NPS/OPS samples and demonstrated over 95% agreement (Cohen's kappa coefficient > 0.95). This study shows the BIONEER NCVM and SCVM assays are comparable with the current standard confirmation assay and are suitable for effective clinical management and control of SARS-CoV-2.

Covid-19 pneumonia: An unusual radiological presentation.

A 26 year old female presented with complaints of high grade fever and cough for 10 days. Nasopharyngeal swab tested for COVID-19 RT-PCR at admission was negative. Clinical examination suggested a patch of bronchial breathing in left infrascapular region and bilateral diffuse rhonchi. Chest X-ray was suggestive of left lower zone consolidation. HRCT showed a large patch of consolidation with GGO along with a cavitary lesion involving left lower lobe. Sputum for RT-PCR COVID 19 was positive. Patient was managed as per covid-19 protocol, subsequently showing clinical and radiological improvement.

The Application of Metagenomic Next-Generation Sequencing in Detection of Pathogen in Bronchoalveolar Lavage Fluid and Sputum Samples of Patients with Pulmonary Infection.

OBJECTIVE: To uncover the application value of metagenomic next-generation sequencing (mNGS) in the detection of pathogen in bronchoalveolar lavage fluid (BALF) and sputum samples. METHODS: Totally, 32 patients with pulmonary infection were included. Pathogens in BALF and sputum samples were tested simultaneously by routine microbial culture and mNGS. Main infected pathogens (bacteria, fungi, and viruses) and their distribution in BALF and sputum samples were analyzed. Moreover, the diagnostic performance of mNGS in paired BALF and sputum samples was assessed. RESULTS: The pathogen culture results were positive in 9 patients and negative in 13 patients. No statistical differences were recorded on the sensitivity (78.94% vs. 63.15%, p = 0.283) and specificity (62.50% vs. 75.00%, p = 0.375) of mNGS diagnosis in bacteria and fungus in two types of samples. As shown in mNGS detection, 10 patients' two samples were both positive, 13 patients' two samples were both negative, 7 patients were only positive in BALF samples, and 2 patients' sputum samples were positive. Main viruses mNGS detected were EB virus, human adenovirus 5, herpes simplex virus type 1, and human cytomegalovirus. Kappa consensus analysis indicated that mNGS showed significant consistency in detecting pathogens in two samples, no matter bacteria (p < 0.001), fungi (p = 0.026), or viruses (p = 0.008). CONCLUSION: mNGS showed no statistical differences in sensitivity and specificity of pathogen detection in BALF and sputum samples. Under certain conditions, sputum samples might be more suitable for pathogen detection because of invasiveness of BALF samples.

Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens.

The rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. In this proof-of-concept study, we explored the potential of targeted mass spectrometry (MS) based proteomics for the detection of SARS-CoV-2 proteins in both research samples and clinical specimens. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) MS in infected Vero E6 cells. For tryptic peptides of Nucleocapsid protein, the limit of detection was estimated to be in the mid-attomole range (9E-13 g). Next, this PRM methodology was applied to the detection of viral proteins in various COVID-19 patient clinical specimens, such as sputum and nasopharyngeal swabs. SARS-CoV-2 proteins were detected in these samples with high sensitivity in all specimens with PCR Ct values <24 and in several samples with higher CT values. A clear relationship was observed between summed MS peak intensities for SARS-CoV-2 proteins and Ct values reflecting the abundance of viral RNA. Taken together, these results suggest that targeted MS based proteomics may have the potential to be used as an additional tool in COVID-19 diagnostics.

Detection of Infectious Viruses Using CRISPR-Cas12-Based Assay.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease-19 (COVID-19), has severely influenced public health and economics. For the detection of SARS-CoV-2, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)-based assays have been emerged because of their simplicity, sensitivity, specificity, and wide applicability. Herein, we have developed a CRISPR-Cas12-based assay for the detection of SARS-CoV-2. In the assay, the target amplicons are produced by isothermal reverse transcription recombinase polymerase amplification (RT-RPA) and recognized by a CRISPR-Cas12a/guide RNA (gRNA) complex that is coupled with the collateral cleavage activity of fluorophore-tagged probes, allowing either a fluorescent measurement or naked-eye detection on a lateral flow paper strip. This assay enables the sensitive detection of SARS-CoV-2 at a low concentration of 10 copies per sample. Moreover, the reliability of the method is verified by using nasal swabs and sputum of COVID-19 patients. We also proved that the current assay can be applied to other viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), with no major changes to the basic scheme of testing. It is anticipated that the CRISPR-Cas12-based assay has the potential to serve as a point-of-care testing (POCT) tool for a wide range of infectious viruses.

Respiratory Viruses in Acute Exacerbations of Bronchiectasis.

BACKGROUND: Bacterial infections are well known factors underlying acute exacerbations in bronchiectasis. However, viral infections may also contribute to acute exacerbations. We aimed to assess the rate of viral detection in acute exacerbations of bronchiectasis, and the associated clinical factors. METHODS: Diagnostic tests for viral and bacterial etiologies were performed in 792 patients with bronchiectasis who visited the emergency room or the respiratory care inpatient unit in a tertiary referral center in South Korea. All patients were diagnosed with bronchiectasis by chest computerized tomography and were prescribed antibiotics for a minimum of 3 days. RESULTS: Viral pathogens were detected in 202 of the 792 enrolled patients (25.5%). The most common viral pathogen isolated was influenza A virus (24.8%), followed by rhinovirus (22.4%), influenza B virus (9.8%), respiratory syncytial virus B (8.9%), and human metapneumovirus (6.1%). In 145 patients, a viral, but not bacterial, pathogen was detected, whereas no pathogens were found in 443 patients with exacerbations. Multivariable analysis revealed that female sex and chronic heart disease as a comorbidity were positively associated with viral detection in acute exacerbations of patients with bronchiectasis, whereas the presence of radiographic infiltration was negatively associated. CONCLUSION: Respiratory viruses were identified in approximately 25% of the acute exacerbations observed among patients with bronchiectasis. Of the viruses detected, influenza viruses and rhinovirus made up over 50%. More attention to viruses as possible causative pathogens for acute deteriorating symptoms in patients with bronchiectasis is warranted.

Detection of SARS-CoV-2 Infection in Gargle, Spit, and Sputum Specimens.

The gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection diagnosis is reverse transcription (RT)-PCR from a nasopharyngeal swab specimen (NPS). Its collection involves close contact between patients and health care workers, requiring a significant amount of workforce and putting them at risk of infection. We evaluated self-collection of alternative specimens and compared their sensitivity and cycle threshold (CT) values to those of NPS. We visited acute coronavirus disease 2019 (COVID-19) outpatients to collect concomitant NPS and gargle specimens and had patients self-collect gargle and either sputum or spit specimens the next morning. We included 40 patients and collected 40 concomitant NPS and gargle specimens, as well as 40 gargle, 22 spit, and 16 sputum specimens the next day (2 patients could not produce sputum). All specimens were as sensitive as NPS. Gargle specimens had a sensitivity of 0.97 (95% confidence interval [CI], 0.92 to 1.00), whether collected concomitantly with NPS or the next morning. Next-morning spit and sputum specimens showed sensitivities of 1.00 (95% CI, 1.00 to 1.00) and 0.94 (95% CI, 0.87 to 1.00]), respectively. The gargle specimens had significantly higher mean CT values of 29.89 (standard deviation [SD], 4.63; P < 0.001) and 29.25 (SD, 3.99; P < 0.001) when collected concomitantly and the next morning, respectively, compared to NPS (22.07 [SD, 4.63]). CT values obtained with spit (23.51 [SD, 4.57]; P = 0.11) and sputum (25.82 [SD, 9.21]; P = 0.28) specimens were close to those of NPS. All alternative specimen collection methods were as sensitive as NPS, but spit collection appeared more promising, with a low CT value and ease of collection. Our findings warrant further investigation. IMPORTANCE Control of the COVID-19 pandemic relies heavily on a test-trace-isolate strategy. The most commonly used specimen for diagnosis of SARS-CoV-2 infection is a nasopharyngeal swab. However, this method is quite uncomfortable for the patient, requires specific equipment (nose swabs and containers), and requires close proximity to health care workers, putting them at risk of infection. Developing alternative sampling strategies could decrease the burden for health care workers, help overcome potential shortages of equipment, and improve acceptability of testing by reducing patient discomfort.

Pooled sputum to optimise the efficiency and utility of rapid, point-of-care molecular SARS-CoV-2 testing.

BACKGROUND: As SARS-CoV-2 testing expands, particularly to widespread asymptomatic testing, high sensitivity point-of-care PCR platforms may optimise potential benefits from pooling multiple patients' samples. METHOD: We tested patients and asymptomatic citizens for SARS-CoV-2, exploring the efficiency and utility of CovidNudge (i) for detection in individuals' sputum (compared to nasopharyngeal swabs), (ii) for detection in pooled sputum samples, and (iii) by modelling roll out scenarios for pooled sputum testing. RESULTS: Across 295 paired samples, we find no difference (p = 0.1236) in signal strength for sputum (mean amplified replicates (MAR) 25.2, standard deviation (SD) 14.2, range 0-60) compared to nasopharyngeal swabs (MAR 27.8, SD 12.4, range 6-56). At 10-sample pool size we find some drop in absolute strength of signal (individual sputum MAR 42.1, SD 11.8, range 13-60 vs. pooled sputum MAR 25.3, SD 14.6, range 1-54; p < 0.0001), but only marginal drop in sensitivity (51/53,96%). We determine a limit of detection of 250 copies/ml for an individual test, rising only four-fold to 1000copies/ml for a 10-sample pool. We find optimal pooled testing efficiency to be a 12-3-1-sample model, yet as prevalence increases, pool size should decrease; at 5% prevalence to maintain a 75% probability of negative first test, 5-sample pools are optimal. CONCLUSION: We describe for the first time the use of sequentially dipped sputum samples for rapid pooled point of care SARS-CoV-2 PCR testing. The potential to screen asymptomatic cohorts rapidly, at the point-of-care, with PCR, offers the potential to quickly identify and isolate positive individuals within a population "bubble".

Field evaluation of a prototype tuberculosis lipoarabinomannan lateral flow assay on HIV-positive and HIV-negative patients.

Detection of tuberculosis at the point-of-care (POC) is limited by the low sensitivity of current commercially available tests. We describe a diagnostic accuracy field evaluation of a prototype urine Tuberculosis Lipoarabinomannan Lateral Flow Assay (TB-LAM LFA) in both HIV-positive and HIV-negative patients using fresh samples with sensitivity and specificity as the measures of accuracy. This prototype combines a proprietary concentration system with a sensitive LFA. In a prospective study of 292 patients with suspected pulmonary tuberculosis in Uganda, the clinical sensitivity and specificity was compared against a microbiological reference standard including sputum Xpert MTB/RIF Ultra and solid and liquid culture. TB-LAM LFA had an overall sensitivity of 60% (95%CI 51-69%) and specificity of 80% (95%CI 73-85%). When comparing HIV-positive (N = 86) and HIV-negative (N = 206) patients, there was no significant difference in sensitivity (sensitivity difference 8%, 95%CI -11% to +24%, p = 0.4351) or specificity (specificity difference -9%, 95%CI -24% to +4%, p = 0.2051). Compared to the commercially available Alere Determine TB-LAM Ag test, the TB-LAM LFA prototype had improved sensitivity in both HIV-negative (difference 49%, 95%CI 37% to 59%, p<0.0001) and HIV-positive patients with CD4+ T-cell counts >200cells/µL (difference 59%, 95%CI 32% to 75%, p = 0.0009). This report is the first to show improved performance of a urine TB LAM test for HIV-negative patients in a high TB burden setting. We also offer potential assay refinement solutions that may further improve sensitivity and specificity.

Is it enough for COVID-19 screening test? Limitation of swab test and general characteristics of mild symptom patients.

The most common method for SARS-CoV-2 testing is throat or nasal swabbing by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. In South Korea, drive-through swab test is used for screening system and community treatment centers (CTCs), which admit and treat confirmed COVID-19 patients with mild symptoms, are being used. This retrospective study was conducted on patients admitted to a CTC on March 6, 2020. A total of 313 patients were admitted. The nasal and throat swabs were collected from the upper respiratory tract, and a sputum test was performed to obtain lower respiratory samples. The positive rate of the first set of test, sputum test was higher than that of the swab test (p = 0.011). In the second set of test, 1 week after the first ones, the rate of positive swab tests was relatively high (p = 0.026). In the first set of test, 66 of 152 (43.4%) patients showed 24-h consecutive negative swab test results, when the sputum test results were considered together, that number fell to 29 patients (19.1%) (p < 0.001). Also, in the second set of test, 63 of 164 (38.4%) patients met the discharge criteria only when the swab test was considered; that number fell to 30 (18.3%) when the sputum test results were also considered (p < 0.001). Using the swab test alone is insufficient for screening test and discharge decision. Patients who may have positive result in the sputum test can be missed.

Detection and viral RNA shedding of SARS-CoV-2 in respiratory specimens relative to symptom onset among COVID-19 patients in Bavaria, Germany.

We assessed severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) reverse transcriptase-polymerase chain reaction (RT-PCR) diagnostic sensitivity and cycle threshold (Ct) values relative to symptom onset in symptomatic coronavirus disease-2019 (COVID-19) patients from Bavaria, Germany, of whom a subset was repeatedly tested. Locally weighted scatterplot smoothing method was used to assess the relationship between symptom onset and Ct-values. Kaplan-Meier plots were used to visualise the empirical probability of detecting viral ribonucleic acid (RNA) over time and estimate the time until clearance of viral RNA among the repeatedly tested patients. Among 721 reported COVID-19 cases, the viral RNA was detected in specimens taken between three days before and up to 48 days after symptom onset. The mean Ct-value was 28.6 (95% confidence interval (CI) 28.2-29.0) with the lowest mean Ct-value (26.2) observed two days after symptom onset. Up to 7 days after symptom onset, the diagnostic sensitivity of the RT-PCR among repeatedly sampled patients (n = 208) remained above 90% and decreased to 50% at day 12 (95% CI 10.5-21.5). Our data provide valuable estimates to optimise the timing of sampling of individuals for SARS-CoV-2 detection. A considerable proportion of specimens sampled before symptom onset had Ct-values comparable with Ct-values after symptom onset, suggesting the probability of presymptomatic transmission.

Proteome Profiling of Recombinant DNase Therapy in Reducing NETs and Aiding Recovery in COVID-19 Patients.

Severe coronavirus disease 2019 (COVID-19) can result in pneumonia and acute respiratory failure. Accumulation of mucus in the airways is a hallmark of the disease and can result in hypoxemia. Here, we show that quantitative proteome analysis of the sputum from severe patients with COVID-19 reveal high levels of neutrophil extracellular trap (NET) components, which was confirmed by microscopy. Extracellular DNA from excessive NET formation can increase sputum viscosity and lead to acute respiratory distress syndrome. Recombinant human DNase (Pulmozyme; Roche) has been shown to be beneficial in reducing sputum viscosity and improve lung function. We treated five patients pwith COVID-19 resenting acute symptoms with clinically approved aerosolized Pulmozyme. No adverse reactions to the drug were seen, and improved oxygen saturation and recovery in all severely ill patients with COVID-19 was observed after therapy. Immunofluorescence and proteome analysis of sputum and blood plasma samples after treatment revealed a marked reduction of NETs and a set of statistically significant proteome changes that indicate reduction of hemorrhage, plasma leakage and inflammation in the airways, and reduced systemic inflammatory state in the blood plasma of patients. Taken together, the results indicate that NETs contribute to acute respiratory failure in COVID-19 and that degrading NETs may reduce dependency on external high-flow oxygen therapy in patients. Targeting NETs using recombinant human DNase may have significant therapeutic implications in COVID-19 disease and warrants further studies.

A review of nasopharyngeal swab and saliva tests for SARS-CoV-2 infection: Disease timelines, relative sensitivities, and test optimization.

Testing is an essential part of containment of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. This review summarizes studies for SARS-CoV-2 infection and testing. Nasopharyngeal samples are best at sensitivity detection, especially in early stages of disease and in asymptomatic individuals. Current swab processing involves a 100- to 1000-fold dilution of the patient sample. Future optimization of testing should focus on using smaller volumes of viral transport media and swab designs to increase comfort and increased viral adhesion.

Comparison between RT-qPCR for SARS-CoV-2 and expanded triage in sputum of symptomatic and asymptomatic COVID-19 subjects in Ecuador.

BACKGROUND: The quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) effectively detects the SARS-COV-2 virus. SARS-CoV-2 Nevertheless, some critical gaps remain in the identification and monitoring of asymptomatic people. METHODS: This retrospective study included 733 asymptomatic and symptomatic COVID-19 subjects, who were submitted to the RT-qPCR test. The objective was to assess the efficacy of an expanded triage of subjects undergoing the RT-qPCR test for SARS-COV-2 to identify the largest possible number of COVID-19 cases in a hospital setting in Ecuador. SARS-CoV-2 Firstly, the sensitivity and specificity as well as the predictive values of an expanded triage method were calculated. In addition, the Kappa coefficient was also determined to assess the concordance between laboratory test results and the expanded triage. RESULTS: Of a total of 733 sputum samples; 229 were RT-qPCR-positive (31.2%) and mortality rate reached 1.2%. Overall sensitivity and specificity were 86.0% (95% confidence interval: 81.0-90.0%) and 37.0% (95% confidence interval: 32.0-41.0%) respectively, with a diagnostic accuracy of 52.0% and a Kappa coefficient of 0.73. An association between the positivity of the test and its performance before 10 days was found. CONCLUSIONS: The clinical sensitivity for COVID-19 detection was within acceptable standards, but the specificity still fell below the values of reference. The lack of symptoms did not always mean to have a negative SARS-COV-2 RT-qPCR test. The expanded triage identified a still unnoticed percentage of asymptomatic subjects showing positive results for the SARS-COV-2 RT-qPCR test. The study also revealed a significant relationship between the number of RT-qPCR-positive cases and the performance of the molecular diagnosis within the first 10 days of COVID-19 in the symptomatic group.

Sequence and phylogentic analysis of MERS-CoV in Saudi Arabia, 2012-2019.

BACKGROUND: The Middle East Respiratory Syndrome-related Coronavirus (MERS-CoV) continues to exist in the Middle East sporadically. Thorough investigations of the evolution of human coronaviruses (HCoVs) are urgently required. In the current study, we studied amplified fragments of ORF1a/b, Spike (S) gene, ORF3/4a, and ORF4b of four human MERS-CoV strains for tracking the evolution of MERS-CoV over time. METHODS: RNA isolated from nasopharyngeal aspirate, sputum, and tracheal swabs/aspirates from hospitalized patients with suspected MERS-CoV infection were analyzed for amplification of nine variable genomic fragments. Sequence comparisons were done using different bioinformatics tools available. RESULTS: Several mutations were identified in ORF1a/b, ORF3/4a and ORF4b, with the highest mutation rates in the S gene. Five codons; 4 in ORF1a and 1 in the S gene, were found to be under selective pressure. Characteristic amino acid changes, potentially hosted and year specific were defined across the S protein and in the receptor-binding domain Phylogenetic analysis using S gene sequence revealed clustering of MERS-CoV strains into three main clades, A, B and C with subdivision of with clade B into B1 to B4. CONCLUSIONS: In conclusion, MERS-CoV appears to continuously evolve. It is recommended that the molecular and pathobiological characteristics of future MERS-CoV strains should be analyzed on regular basis to prevent potential future outbreaks at early phases.