Search

Objective: To study the frequency of microbiological etiology of respiratory infections in patients with long COVID and their associated clinical and radiological findings., Methods: Nasopharyngeal swabs and sputum specimens were collected from 97 patients with respiratory illness stemming from long COVID. The specimens were assessed for their microbiological profile (bacteria and virus) and their association with the overall clinical and radiological picture., Results: In total, 23 (24%) patients with long COVID had viral infection (n = 12), bacterial infection (n = 9), or coinfection (n = 2). Microorganisms were detected at significantly higher rates in hospitalized patients, patients with moderate COVID-19, and patients with asthma (P < .05). Tachycardia (65%) was the most common symptom at presentation. A statistically significant number of patients with long COVID who had viral infection presented with cough and myalgia; and a statistically significant number of patients with long COVID who had bacterial infection presented with productive coughing (P < .05). Post-COVID fibrotic changes were found in 61% of cohort patients (31/51)., Conclusion: A decreasing trend of respiratory pathogens (enveloped viruses and bacteria) was found in long COVID. An analysis including a larger group of viral- or bacterial-infected patients with long COVID is needed to obtain high-level evidence on the presenting symptoms (cough, myalgia) and their association with the underlying comorbidities and severity., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Exacerbations are implicated in bronchiectasis and COPD, which frequently co-exist [COPD-Bronchiectasis association (CBA)]. We aimed to determine the bacterial and viral spectrum at stable-state and exacerbation onset of CBA, and their association with exacerbations and clinical outcomes of CBA as compared with bronchiectasis., Methods: We prospectively collected spontaneous sputum from adults with CBA, bronchiectasis with (BO) and without airflow obstruction (BNO) for bacterial culture and viral detection at stable-state and exacerbations., Results: We enrolled 76 patients with CBA, 58 with BO, and 138 with BNO (711 stable and 207 exacerbation visits). Bacterial detection rate increased from BNO, CBA to BO at steady-state (P = 0.02), but not at AE onset (P = 0.91). No significant differences in viral detection rate were found among BNO, CBA and BO. Compared with steady-state, viral isolations occurred more frequently at exacerbation in BNO (15.8 % vs 32.1 %, P = 0.001) and CBA (19.5 % vs 30.6 %, P = 0.036) only. In CBA, isolation of viruses, human metapneumovirus and bacteria plus viruses was associated with exacerbation. Repeated detection of Pseudomonas aeruginosa (PA) correlated with higher modified Reiff score (P = 0.032) in CBA but not in BO (P = 0.178). Repeated detection of PA yielded a shorter time to the first exacerbation in CBA [median: 4.3 vs 11.1 months, P = 0.006] but not in BO (median: 8.4 vs 7.6 months, P = 0.47)., Conclusions: Isolation of any viruses, human metapneumovirus and bacterialplus viruses was associated with CBA exacerbations. Repeated detection of PA confers greater impact of future exacerbations on CBA than on BO., Competing Interests: Declaration of competing interest The authors declared no conflict of interest with any financial organization regarding the material discussed in the manuscript., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Metagenomic next-generation sequencing (mNGS) technology has been widely used to diagnose various infections. Based on the most common pathogen profiles, targeted mNGS (tNGS) using multiplex PCR has been developed to detect pathogens with predesigned primers in the panel, significantly improving sensitivity and reducing economic burden on patients. However, there are few studies on summarizing pathogen profiles of pulmonary infections in immunocompetent and immunocompromised patients in Jilin Province of China on large scale., Methods: From January 2021 to December 2023, bronchoalveolar lavage fluid (BALF) or sputum samples from 546 immunocompetent and immunocompromised patients with suspected community-acquired pneumonia were collected. Pathogen profiles in those patients on whom mNGS was performed were summarized. Additionally, we also evaluated the performance of tNGS in diagnosing pulmonary infections., Results: Combined with results of mNGS and culture, we found that the most common bacterial pathogens were Pseudomonas aeruginosa , Klebsiella pneumoniae , and Acinetobacter baumannii in both immunocompromised and immunocompetent patients with high detection rates of Staphylococcus aureus and Enterococcus faecium , respectively. For fungal pathogens, Pneumocystis jirovecii was commonly detected in patients, while fungal infections in immunocompetent patients were mainly caused by Candida albicans . Most of viral infections in patients were caused by Human betaherpesvirus 5 and Human gammaherpesvirus 4. It is worth noting that, compared with immunocompetent patients (34.9%, 76/218), more mixed infections were found in immunocompromised patients (37.8%, 14/37). Additionally, taking final comprehensive clinical diagnoses as reference standard, total coincidence rate of BALF tNGS (81.4%, 48/59) was much higher than that of BALF mNGS (40.0%, 112/280)., Conclusions: Our findings supplemented and classified the pathogen profiles of pulmonary infections in immunocompetent and immunocompromised patients in Jilin Province of China. Most importantly, our findings can accelerate the development and design of tNGS specifically used for regional pulmonary infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liu, Wu, Xiao, Zou, Hao and Jiang.)

Objectives: To evaluate diagnostic yield and feasibility of integrating testing for TB and COVID-19 using molecular and radiological screening tools during community-based active case-finding (ACF)., Methods: Community-based participants with presumed TB and/or COVID-19 were recruited using a mobile clinic. Participants underwent simultaneous point-of-care (POC) testing for TB (sputum; Xpert Ultra) and COVID-19 (nasopharyngeal swabs; Xpert SARS-CoV-2). Sputum culture and SARS-CoV-2 RT-PCR served as reference standards. Participants underwent ultra-portable POC chest radiography with computer-aided detection (CAD). TB infectiousness was evaluated using smear microscopy, cough aerosol sampling studies (CASS), and chest radiographic cavity detection. Feasibility of POC testing was evaluated via user-appraisals., Results: Six hundred and one participants were enrolled, with 144/601 (24.0%) reporting symptoms suggestive of TB and/or COVID-19. 16/144 (11.1%) participants tested positive for TB, while 10/144 (6.9%) tested positive for COVID-19 (2/144 [1.4%] had concurrent TB/COVID-19). Seven (7/16 [43.8%]) individuals with TB were probably infectious. Test-specific sensitivity and specificity (95% CI) were: Xpert Ultra 75.0% (42.8-94.5) and 96.9% (92.4-99.2); Xpert SARS-CoV-2 66.7% (22.3-95.7) and 97.1% (92.7-99.2). Area under the curve (AUC) for CAD4TB was 0.90 (0.82-0.97). User appraisals indicated POC Xpert to have 'good' user-friendliness., Conclusions: Integrating TB/COVID-19 screening during community-based ACF using POC molecular and radiological tools is feasible, has a high diagnostic yield, and can identity probably infectious persons., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: The performance of the new Respiratory Pathogen panel (fluorescent probe melting curve, FPMC) for the qualitative detection of 12 organisms (chlamydia pneumoniae, mycoplasma pneumoniae, adenovirus, influenza A virus, influenza B virus, parainfluenza virus, rhinovirus, etc.) was assessed., Methods: Prospectively collected nasopharyngeal swab (NPS) and sputum specimens (n = 635) were detected by using the FPMC panel, with the Sanger sequencing method as the comparative method., Results: The overall percent concordance between the FPMC analysis method and the Sanger sequencing method was 100% and 99.66% for NPS and sputum specimens, respectively. The FPMC testified an overall positive percent concordance of 100% for both NPS and sputum specimens. The FPMC analysis method also testified an overall negative percent concordance of 100% and 99.38% for NPS and sputum specimens, respectively., Conclusions: The FPMC analysis method is a stable and accurate assay for rapid, comprehensive detecting for respiratory pathogens., (© 2024. The Author(s).)

In recent years, the avian influenza virus has emerged as a significant threat to both human and public health. This study focuses on a patient infected with the H10N3 subtype of avian influenza virus, admitted to the Third People's Hospital of Kunming City on March 6, 2024. Metagenomic RNA sequencing and polymerase chain reaction (PCR) analysis were conducted on the patient's sputum, confirming the H10N3 infection. The patient presented severe pneumonia symptoms such as fever, expectoration, chest tightness, shortness of breath, and cough. Phylogenetic analysis of the Haemagglutinin (HA) and neuraminidase (NA) genes of the virus showed that the virus was most closely related to a case of human infection with the H10N3 subtype of avian influenza virus found in Zhejiang Province, China. Analysis of amino acid mutation sites identified four mutations potentially hazardous to human health. Consequently, this underscores the importance of continuous and vigilant monitoring of the dynamics surrounding the H10N3 subtype of avian influenza virus, utilizing advanced genomic surveillance techniques., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dai, Zhao, Xia, Zhang, Ding, Li, Hou, Xiong, Jian, Liu and Liu.)

Background: Respiratory viral infections are major drivers of chronic obstructive pulmonary disease (COPD) exacerbations. Interferon-β is naturally produced in response to viral infection, limiting replication. This exploratory study aimed to demonstrate proof-of-mechanism, and evaluate the efficacy and safety of inhaled recombinant interferon-β1a (SNG001) in COPD. Part 1 assessed the effects of SNG001 on induced sputum antiviral interferon-stimulated gene expression, sputum differential cell count, and respiratory function. Part 2 compared SNG001 and placebo on clinical efficacy, sputum and serum biomarkers, and viral clearance., Methods: In Part 1, patients (N = 13) with stable COPD were randomised 4:1 to SNG001 or placebo once-daily for three days. In Part 2, patients (N = 109) with worsening symptoms and a positive respiratory viral test were randomised 1:1 to SNG001 or placebo once-daily for 14 days in two Groups: A (no moderate exacerbation); B (moderate COPD exacerbation [i.e., acute worsening of respiratory symptoms treated with antibiotics and/or oral corticosteroids])., Results: In Part 1, SNG001 upregulated sputum interferon gene expression. In Part 2, there were minimal SNG001-placebo differences in the efficacy endpoints; however, whereas gene expression was initially upregulated by viral infection, then declined on placebo, levels were maintained with SNG001. Furthermore, the proportion of patients with detectable rhinovirus (the most common virus) on Day 7 was lower with SNG001. In Group B, serum C-reactive protein and the proportion of patients with purulent sputum increased with placebo (suggesting bacterial infection), but not with SNG001. The overall adverse event incidence was similar with both treatments., Conclusions: Overall, SNG001 was well-tolerated in patients with COPD, and upregulated lung antiviral defences to accelerate viral clearance. These findings warrant further investigation in a larger study., Trial Registration: EU clinical trials register (2017-003679-75), 6 October 2017., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist.

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 (C t ) 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.

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., Competing Interests: The authors have declared that no competing interests exist.

Without an effective vaccine against SARS-CoV-2, the build-up of herd immunity through natural infection has been suggested as a means to control COVID-19. Although population immunity is typically estimated by the serological investigation of recovered patients, humoral immunity in asymptomatic subjects has not been well studied, although they represent a large proportion of all SARS-CoV-2 infection cases. In this study, we conducted a serosurvey of asymptomatic infections among food workers and performed serological and cellular response analyses of asymptomatic subjects in Wuhan, the original epicenter of the COVID-19 outbreak. Our data showed that up to 5.91% of the food workers carried SARS-CoV-2 IgG antibodies asymptomatically; however, in 90.4% of them, the antibody level declined over a 2-week period. IgM and IgG antibodies, including neutralizing antibodies, were significantly lower in asymptomatic subjects than in recovered symptomatic patients with similar disease courses. Furthermore, the asymptomatic subjects showed lymphopenia and a prominent decrease in the B-cell population, as well as a low frequency of antibody-secreting cells and a low cytokine response. These factors probably contributed to the low and unsustained antibody levels in asymptomatic subjects. Our results show that asymptomatic subjects are likely to be vulnerable to SARS-CoV-2 reinfection, and neither the proportion of population immunity nor the breadth of immune responses is sufficient for herd immunity.

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wanghui Shi and Shanshan Zhu.)

Background: Although some respiratory virus infections are known to contribute to the development and exacerbation of asthma, commensal viromes in airway have not been extensively studied due to technical challenges., Objectives: This study investigated the characteristics of the virome in asthmatic airways., Methods: Both the bacteriome and virome profiles in sputum from 12 healthy individuals, 15 patients with nonsevere asthma, and 15 patients with severe asthma were analyzed and assessed for the association with clinical characteristics such as severity, exacerbation, Asthma Control Test (ACT), and lung function., Results: While analysis of the 16S ribosomal RNA bacteriome in the airway showed no differences, clear contrasts in the diversity and composition of airway viromes were observed between healthy controls and patients with asthma. Herpesviruses were the most abundant type of virus in the asthma group (44.6 ± 4.6%), mainly with cytomegalovirus (CMV) and EBV accounting for 24.5 ± 3.3% and 16.9 ± 3.5%, respectively, in contrast to those in the healthy controls (5.4 ± 2.5% and 7.1 ± 3.0%, respectively). CMV and EBV were more abundant in patients with asthma who experienced exacerbation, and their abundance showed correlation with more severe asthma, lower ACT score, and lower lung function. On the contrary, bacteriophage that is abundant in healthy controls was severely reduced in patients with asthma in the order of nonsevere and severe asthma and presented significant positive correlation with ACT and FEV 1 /forced vital capacity., Conclusions: Lung viromes, especially, CMV, EBV, and bacteriophage may be potential biomarkers of asthma severity and exacerbation., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Conflicts of interest The authors have none to declare., (Copyright © 2021 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.)

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 ( C T ) 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 C T 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]). C T 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 C T 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.

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., (© 2021 Wiley Periodicals LLC.)

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., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2021 The Korean Academy of Medical Sciences.)

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.

Background: Prolonged viral RNA detection in respiratory samples from patients with COVID-19 has been described, but the clinical relevance remains unclear. We studied the dynamics of SARS-CoV-2 on a group and individual level in intubated ICU patients., Methods: In a cohort of 86 patients, we analysed SARS-CoV-2 RT-PCR results on nasopharyngeal and sputum samples (obtained as part of clinical care twice a week) according to time after intubation. Subsequently, we performed survival analyses., Results: 870 samples were tested by RT-PCR. Overall viral load was highest in the first week (median nasopharynx 3.5, IQR 1.5-4.3; median sputum 4.3, IQR 3.3-5.6) and decreased over time. In 20% of patients a relapsing pattern was observed. Nasopharyngeal and sputum PCR status on day 14 was not significantly associated with survival up to day 60 in this small cohort., Conclusion: In general SARS-CoV-2 RNA levels in respiratory samples in patients with severe COVID-19 decrease after the first week after intubation, but individual SARS-CoV-2 RNA levels can show a relapsing pattern. Larger studies are needed to address the association of clearance of SARS-CoV-2 RNA from respiratory samples with survival, because we observed a trend towards better survival in patients with early clearance from sputum., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: HR, LI, and BH are employees of Biopromic AB. Biopromic AB is developing a point-of-care TB LAM test similar to the one presented. JTC, BDG, AB, BBL, HVH, VMH, SB, BHW, CB, DB, and AS were employees of Global Good Fund, Intellectual Ventures. BH is also an author on patents in the field of TB immunoassays, “Monoclonal antibody, method, kit and use” (WO2016012449) and “Method for removing inhibitory components” (US20190302115). JTC, BDG, and BHW are authors on a patent in the field of analyte concentration devices, “Device for rapid detection of tuberculosis-lipoarabinomannan (TB-LAM) with enhanced sensitivity” (US10830760). AP is an author on patents in the field of mycobacteria detection, “Novel anti-lam and anti-pim6/lam monoclonal antibodies for diagnosis and treatment of mycobacterium tuberculosis infections” (US20190038747) and “Methods for dual detection and differentiation of infection by mycobactierum tuberculosis complex and nontuberculous mycobacteria” (WO2020018806). None of the disclosed competing interest alter our adherence to PLOS ONE policies on sharing data and materials.

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".

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.

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.

Objective: The real-time polymerase chain reaction (RT-PCR) test is recommended for the diagnosis of COVID-19 and provides a powerful tool to identify new infections and facilitate contact tracing. In fact, as the prevalence of COVID-19 decreases, this RT-PCR testing remains as the main preventive measure to avoid rebound. However, inconsistent results can lead to misdiagnoses in the clinic. These inconsistencies are due to the variability in (1) the collection times of biological samples post infection, and (2) sampling procedures., Methods: We applied the Kaplan-Meier method and multivariate logistic regression on RT-PCR results from 258 confirmed patients with COVID-19 to evaluate the factors associated with negative conversion. We also estimated the proportion (%) of negative conversion among patients who had tested twice or more, and compared the proportions arising from oropharyngeal swabs, sputum, and combined double testing, respectively., Main Results: The proportion of negative conversion was 6.7% on day 4, 16.4% on day 7, 41.0% at 2 weeks, and 61.0% at 3 weeks post-admission. We also found that 34.1% and 60.3% of subjects had at least one negative RT-PCR result on days 7 and 14 after the onset of symptoms, respectively. The proportion of negative conversions following sputum testing was higher than that from oropharyngeal swabs in the early stages but this declined after the onset of symptoms., Conclusion: In the absence of effective treatments or vaccines, efficient testing strategies are critical if we are to control the COVID-19 epidemic. According to this study, early, consecutive and combined double testing, will be the key to identify infected patients, particularly for asymptomatic and mild symptomatic cases. These strategies will minimize misdiagnosis and the ineffective isolation of infected patients., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic. The understanding of the transmission and the duration of viral shedding in SARS-CoV-2 infection is still limited., Objectives: To assess the timeframe and potential risk of SARS-CoV-2 transmission from hospitalized COVID-19 patients in relation to antibody response., Method: We performed a cross-sectional study of 36 COVID-19 patients hospitalized at Karolinska University Hospital. Patients with more than 8 days of symptom duration were sampled from airways, for PCR analysis of SARS-CoV-2 RNA and in vitro culture of replicating virus. Serum SARS-CoV-2-specific immunoglobulin G (IgG) and neutralizing antibodies titers were assessed by immunofluorescence assay (IFA) and microneutralization assay., Results: SARS-CoV-2 RNA was detected in airway samples in 23 patients (symptom duration median 15 days, range 9-53 days), whereas 13 patients were SARS-CoV-2 RNA negative (symptom duration median 21 days, range 10-37 days). Replicating virus was detected in samples from 4 patients at 9-16 days. All but two patients had detectable levels of SARS-CoV-2-specific IgG in serum, and SARS-CoV-2 neutralizing antibodies were detected in 33 out of 36 patients. Total SARS-CoV-2-specific IgG titers and neutralizing antibody titers were positively correlated. High levels of both total IgG and neutralizing antibody titers were observed in patients sampled later after symptom onset and in patients where replicating virus could not be detected., Conclusions: Our data suggest that the presence of SARS-Cov-2 specific antibodies in serum may indicate a lower risk of shedding infectious SARS-CoV-2 by hospitalized COVID-19 patients.

Viruses are important agents in lung function deterioration in Cystic Fibrosis (CF). To date, no standard operating procedures (SOPs) have been established to determine which sampling method is the most effective for an optimal virological diagnosis of respiratory viral infections in CF. Here we investigated the performances of two sampling sites, sputum samples versus nasopharyngeal (NP) swabs, for thirty participants from three CF centres presenting an acute respiratory infection. Sputum and NP samples were simultaneously collected and multiplex PCR targeting 16 to 18 viruses were performed. Viruses were detected for 18/30 patients (60%). A high concordance between the sputum and NP samples was observed in 25 (83%) paired samples of which 13 tested positive and 12 tested negative. These results highlighted the relevance of sputum sampling for diagnostic of respiratory viruses in CF, which is less invasive and better accepted by CF patients than NP, and allows accurate bacterial detection., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020. Published by Elsevier B.V.)

The novel human coronavirus SARS-CoV-2 has been responsible for a worldwide pandemic. Although media transmission through contaminated surfaces is one of the most recognized ways of transmission, the study on the number and viability of viruses surviving on a surface after leaving the host represents a "blind spot" in current research. In this paper we have reviewed studies on the physical process of droplet evaporation on media surfaces, and analyzed the recent literature related to experiments on the decay of the viral concentration and infectious activity of SARS-CoV-2 and other viruses on those surface and in the air. The huge differences in the risk of media transmission of large saliva and sputum droplets were analyzed in terms of time elapsed. Due to the rapid decrease of water content in the evaporated droplets and the increased concentration of each component, the living environment of the virus tended to deteriorate sharply, and virus concentration plummeted within a few minutes. Although a virus can be detected in a matter of hours, tens of hours, or days, the risk of transmission is negligible compared to when it first left the host. This study suggests that the key to prevention and control is to start from the source, the earlier the better. It is extremely important to develop good public health habits, wear masks, and wash hands frequently. That said, excessive disinfection and sterilization of surfaces during a later period may have adverse effects., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

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.

Background: The novel coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), continues to remain a global challenge. There is emerging evidence of SARS-CoV-2 virus found in the blood of patients from China and some developed countries. However, there is inadequate data reported in Ghana and other parts of Africa, where blood transfusion service heavily relies on voluntary and replacement blood donors. This study aimed to investigate whether plasma of infected individuals could pose significant transfusion transmitted risk of COVID-19 in Ghanaian populations., Methods: This cross-sectional retrospective study was conducted at the Kumasi Centre for Collaborative Research into Tropical Medicine (KCCR), KNUST, Ghana. Study subjects comprised contacts of COVID-19 individuals, those with classical symptoms of COVID-19 and individuals who had recovered based on the new Ghana discharge criteria. Whole blood, sputum or deep coughed saliva samples were collected and transported to KCCR for SARS-CoV-2 testing. Viral nucleic acid was extracted from sputum/nasopharyngeal samples using Da An Gene column based kit and from plasma using LBP nucleic acid extraction kit. Real-Time PCR was performed specifically targeting the ORF1ab and Nucleocapsid (N) genomic regions of the virus., Results: A total of 97 individuals were recruited into the study, with more than half being males (58; 59.7%). The mean age of all subjects was 33 years (SD = 7.7) with minimum being 22 years and maximum 56 years. Majority (76; 78.4%) of all the subjects were asymptomatic, and among the few symptomatic subjects, cough (10; 10.3%) was the most predominant symptom. Of the 97 sputum samples tested, 79 (81.4%) were positive for SARS-CoV-2. We identified SARS-CoV-2 viral RNA in the plasma of 1 (1.03%) subject who had clinically recovered., Conclusion: This study reports the identification of SARS-CoV-2 viral RNA in a convalescent individual in Ghana. Due to the low prevalence observed and the marginal cycling thresholds associated, the risk of transfusion transmission of SARS-CoV-2 is negligible. Well-powered studies and advanced diagnostics to determine infectious viremia is recommended to further evaluate the potential risk of hematogenous transmission among recovered patients., Competing Interests: The authors have declared that no competing interests exist.

Background: An appropriate specimen is of paramount importance in Real Time reverse transcription-polymerase chain reaction (rRT-PCR) based diagnosis of novel coronavirus (nCoV) disease (COVID-19). Thus, it's pertinent to evaluate various diversified clinical specimens' diagnostic utility in both diagnosis and follow-up of COVID-19., Methods: A total of 924 initial specimens from 130 COVID-19 symptomatic cases before initiation of treatment and 665 follow up specimens from 15 randomly selected cases comprising of equal number of nasopharyngeal swab (NPS), oropharyngeal swab (OPS), combined NPS and OPS (Combined swab), sputum, plasma, serum and urine were evaluated by rRT-PCR., Results: Demographic analysis showed males (86) twice more affected by COVID-19 than females (44) (p = 0.00001). Combined swabs showed a positivity rate of 100% followed by NPS (91.5%), OPS (72.3%), sputum (63%), while nCoV was found undetected in urine, plasma and serum specimens. The lowest cycle threshold (Ct) values of targeted genes E, ORF1b and RdRP are 10.56, 10.14 and 12.26 respectively and their lowest average Ct values were found in combined swab which indicates high viral load in combined swab among all other specimen types. Analysis of 665 follow-up multi-varied specimens also showed combined swab as the last specimen among all specimen types to become negative, after an average 6.6 (range 4-10) days post-treatment, having lowest (15.48) and average (29.96) Ct values of ORF1b respectively indicating posterior nasopharyngeal tract as primary nCoV afflicted site with high viral load., Conclusion: The combined swab may be recommended as a more appropriate specimen for both diagnosis and monitoring of COVID-19 treatment by rRT-PCR for assessing virus clearance to help physicians in taking evidence-based decision before discharging patients. Implementing combined swabs globally will definitely help in management and control of the pandemic, as it is the need of the hour., Competing Interests: The authors have declared that no competing interests exist.

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.

Real-time reverse transcription polymerase chain reaction (RT-PCR) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are occasionally repeated when clinicians suspect false-negative results, but the conditions under which repeated RT-PCR testing is warranted remain unclear. We evaluated the practice of repeat RT-PCR testing for SARS-CoV-2 in 45 patients who were retested after an initial negative PCR test. Of these, the diagnosis of coronavirus disease (COVID-19) was confirmed in four patients with typical chest computed tomography (CT) findings and in one patient without typical CT findings in whom the test result was strongly suspected to be false-positive. We recommend repeat RT-PCR testing only for patients with typical CT findings of COVID-19.

How human respiratory physiology and the transport phenomena associated with inhaled airflow in the upper airway proceed to impact transmission of SARS-CoV-2, leading to the initial infection, stays an open question. An answer can help determine the susceptibility of an individual on exposure to a COVID-2019 carrier and can also provide a preliminary projection of the still-unknown infectious dose for the disease. Computational fluid mechanics enabled tracking of respiratory transport in medical imaging-based anatomic domains shows that the regional deposition of virus-laden inhaled droplets at the initial nasopharyngeal infection site peaks for the droplet size range of approximately 2.5-19 [Formula: see text]. Through integrating the numerical findings on inhaled transmission with sputum assessment data from hospitalized COVID-19 patients and earlier measurements of ejecta size distribution generated during regular speech, this study further reveals that the number of virions that may go on to establish the SARS-CoV-2 infection in a subject could merely be in the order of hundreds.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease 2019 (COVID-19) continue to impact countries worldwide. At present, inadequate diagnosis and unreliable evaluation systems hinder the implementation and development of effective prevention and treatment strategies. Here, we conducted a horizontal and longitudinal study comparing the detection rates of SARS-CoV-2 nucleic acid in different types of samples collected from COVID-19 patients and SARS-CoV-2-infected monkeys. We also detected anti-SARS-CoV-2 antibodies in the above clinical and animal model samples to identify a reliable approach for the accurate diagnosis of SARS-CoV-2 infection. Results showed that, regardless of clinical symptoms, the highest detection levels of viral nucleic acid were found in sputum and tracheal brush samples, resulting in a high and stable diagnosis rate. Anti-SARS-CoV-2 immunoglobulin M (IgM) and G (IgG) antibodies were not detected in 6.90% of COVID-19 patients. Furthermore, integration of nucleic acid detection results from the various sample types did not improve the diagnosis rate. Moreover, dynamic changes in SARS-CoV-2 viral load were more obvious in sputum and tracheal brushes than in nasal and throat swabs. Thus, SARS-CoV-2 nucleic acid detection in sputum and tracheal brushes was the least affected by infection route, disease progression, and individual differences. Therefore, SARS-CoV-2 nucleic acid detection using lower respiratory tract samples alone is reliable for COVID-19 diagnosis and study.

A voltammetric genosensor has been developed for the early diagnosis of COVID-19 by determination of RNA-dependent RNA polymerase (RdRP) sequence as a specific target of novel coronavirus. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) uses an RdRP for the replication of its genome and the transcription of its genes. Here, the silver ions (Ag + ) in the hexathia-18-crown-6 (HT18C6) were used for the first time as a redox probe. Then, the HT18C6(Ag) incorporated carbon paste electrode (CPE) was further modified with chitosan and PAMAM dendrimer-coated silicon quantum dots (SiQDs@PAMAM) for immobilization of probe sequences (aminated oligonucleotides). The current intensity of differential pulse voltammetry using the redox probe was found to decrease with increasing the concentration of target sequence. Based on such signal-off trend, the proposed genosensor exhibited a good linear response to SARS-CoV-2 RdRP in the concentration range 1.0 pM-8.0 nM with a regression equation I (μA) = - 6.555 log [RdRP sequence] (pM) + 32.676 (R 2  = 0.995) and a limit of detection (LOD) of 0.3 pM. The standard addition method with different spike concentrations of RdRP sequence in human sputum samples showed a good recovery for real sample analysis (> 95%). Therefore, the developed voltammetric genosensor can be used to determine SARS-CoV-2 RdRP sequence in sputum samples. PAMAM-functionalized SiQDs were used as a versatile electrochemical platform for the SARS-CoV-2 RdRP detection based on a signal off sensing strategy. In this study, for the first time, the silver ions (Ag + ) in the hexathia-18-crown-6 carrier were applied as an electrochemical probe.

Our study intended to longitudinally explore the prediction effect of immunoglobulin A (IgA) on pulmonary exudation progression in COVID-19 patients. The serum IgA was tested with chemiluminescence method. Autoregressive moving average model was used to extrapolate the IgA levels before hospital admission. The positive rate of IgA and IgG in our cohort was 97% and 79.0%, respectively. In this study, the IgA levels peaks within 10-15 days after admission, while the IgG levels peaks at admission. We found that the time difference between their peaks was about 10 days. Viral RNA detection results showed that the positive rate in sputum and feces were the highest. Blood gas analysis showed that deterioration of hypoxia with the enlargement of pulmonary exudation area. And alveolar-arterial oxygen difference and oxygenation index were correlated with IgA and IgG. The results of biopsy showed that the epithelium of lung was exfoliated and the mucosa was edematous. In severe COVID-19 patients, the combination of IgA and IgG can predict the progress of pulmonary lesions and is closely related to hypoxemia and both also play an important defense role in invasion and destruction of bronchial and alveolar epithelium by SARS-CoV-2., (© 2020 Wiley Periodicals LLC.)

Background: Although most observational studies identify viral or bacterial pathogens in 50% or less of patients hospitalized with community-acquired pneumonia (CAP), we previously demonstrated that a multi-test bundle (MTB) detected a potential pathogen in 73% of patients. This study compares detection rates for potential pathogens with the MTB versus the Biofire® Pneumonia FilmArray® panel (BPFA) multiplex PCR platform and presents an approach for integrating BPFA results as a foundation for subsequent antibiotic stewardship (AS) activities., Methods: Between January 2017 to March 2018, all patients admitted for CAP were enrolled. Patients were considered evaluable if all elements of the MTB and the BPFA were completed, and they met other a priori inclusion criteria. The primary endpoint was the percentage of potential pathogens detected using the MTB (8 viral and 6 bacterial targets) versus the BPFA (8 viral and 18 bacterial targets). Blood and sputum cultures were performed on all patients. Two or more procalcitonin (PCT) levels assisted clinical assessments as to whether detected bacteria were invading or colonizing., Results: Of 585 enrolled patients, 274 were evaluable. A potential viral pathogen was detected in 40.5% with MTB versus 60.9% of patients with BPFA with an odds ratio (95% CI) of 9.00 (4.12 to 23.30) p<0.01. A potential bacterial pathogen was identified in 66.4% with the MTB vs 75.5% with the BPFA odds ratio (95% CI) of 2.09 (1.24 to 3.59), p 0.003). Low PCT levels helped identify detected bacteria as colonizers., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Metagenomic next-generation sequencing (mNGS) holds promise as a diagnostic tool for unbiased pathogen identification and precision medicine. However, its medical utility depends largely on assay simplicity and reproducibility. In the current study, we aimed to develop a streamlined Illumina and Oxford Nanopore-based DNA/RNA library preparation protocol and rapid data analysis pipeline. The Illumina sequencing-based mNGS method was first developed and evaluated using a set of samples with known aetiology. Its sensitivity for RNA viruses (influenza A, H1N1) was < 6.4 × 10 2 EID50/mL, and a good correlation between viral loads and mapped reads was observed. Then, the rapid turnaround time of Nanopore sequencing was tested by sequencing influenza A virus and adenoviruses. Furthermore, 11 respiratory swabs or sputum samples pre-tested for a panel of pathogens were analysed, and the pathogens identified by Illumina sequencing showed 81.8% concordance with qPCR results. Additional sequencing of cerebrospinal fluid (CSF) samples from HIV-1-positive patients with meningitis/encephalitis detected HIV-1 RNA and Toxoplasma gondii sequences. In conclusion, we have developed a simplified protocol that realizes efficient metagenomic sequencing of a variety of clinical samples and pathogen identification in a clinically meaningful time frame.

An epidemic caused by an outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China in December 2019 has since rapidly spread internationally, requiring urgent response from the clinical diagnostics community. We present a detailed overview of the clinical validation and implementation of the first laboratory-developed real-time RT-PCR test offered in the NewYork-Presbyterian Hospital system following the Emergency Use Authorization issued by the US Food and Drug Administration. Nasopharyngeal and sputum specimens (n = 174) were validated using newly designed dual-target real-time RT-PCR (altona RealStar SARS-CoV-2 Reagent) for detecting SARS-CoV-2 in upper respiratory tract and lower respiratory tract specimens. Accuracy testing demonstrated excellent assay agreement between expected and observed values and comparable diagnostic performance to reference tests. The limit of detection was 2.7 and 23.0 gene copies per reaction for nasopharyngeal and sputum specimens, respectively. Retrospective analysis of 1694 upper respiratory tract specimens from 1571 patients revealed increased positivity in older patients and males compared with females, and an increasing positivity rate from approximately 20% at the start of testing to 50% at the end of testing 3 weeks later. Herein, we demonstrate that the assay accurately and sensitively identifies SARS-CoV-2 in multiple specimen types in the clinical setting and summarize clinical data from early in the epidemic in New York City., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Testing is one of the commendable measures for curbing the spread of coronavirus disease (COVID-19). But, it should be done using the most appropriate specimen and an accurate diagnostic test such as real-time reverse transcription-polymerase chain reaction (qRT-PCR). Therefore, a systematic review was conducted to determine the positive detection rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in different clinical specimens using qRT-PCR. A total of 8136 pooled clinical specimens were analyzed to detect SARS-CoV-2, the majority were nasopharyngeal swabs (69.6%). A lower respiratory tract (LRT) specimens had a positive rate (PR) of 71.3% (95% confidence interval [CI]: 60.3%-82.3%) while no virus was detected in the urinogenital specimens. Bronchoalveolar lavage fluid (BLF) specimen had the PR of 91.8% (95% CI: 79.9%-103.7%), followed by rectal swabs; 87.8% (95% CI: 78.6%-96.9%) then sputum; 68.1% (95% CI: 56.9%-79.4%). A low PR was observed in oropharyngeal swabs; 7.6% (95% CI: 5.7%-9.6%) and blood samples; 1.0% (95% CI: -0.1%-2.1%) whereas no SARS-CoV-2 was detected in urine samples. Feces had a PR of 32.8% (95% CI:1 5.8%-49.8%). Nasopharyngeal swab, a widely used specimen had a PR of 45.5% (95% CI: 31.2%-59.7%). In this study, SARS-CoV-2 was highly detected in LRT specimens while no virus was detected in urinogenital specimens. BLF had the highest PR followed by rectal swab then sputum. Nasopharyngeal swab which is widely used had moderate PR. Low PR was recorded in oropharyngeal swab and blood samples while no virus was found in urine samples. Last, the virus was detected in feces, suggesting SARS-CoV-2 transmission by the fecal route., (© 2020 Wiley Periodicals LLC.)

Following the announcement of the first coronavirus disease 2019 (COVID-19) case on 11 March 2020 in Turkey, we aimed to report the coinfection rates, and the clinical, laboratory, radiological distinctive features of viral pneumonia caused by viruses other than severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A cross-sectional study was conducted between 18 and 31 March 2020. COVID-19 suspected cases admitted to pandemic policlinic, who had nasopharyngeal swab specimens tested for both SARS-CoV-2 and other respiratory viral pathogens, were included. Among 112 patients, SARS-CoV-2 was detected in 34 patients (30%). Among the non-SARS-CoV-2 viruses (n = 25, 22%), metapneumovirus (n = 10) was the most frequent agent. There were two coinfections with SARS-CoV-2. Sputum was less in the SARS-CoV-2 group (P = .003). The leukocyte, lymphocyte, and thrombocyte count and C-reactive protein levels were the lowest in the SARS-CoV-2 group (P < .001, P = .04, P < .001, P = .007, respectively). Peripheral involvement (80% vs 20%; P ≤ .001), pure ground-glass opacity (65% vs 33%; P = .04), apicobasal gradient (60% vs 40%; P = .08), involvement of greater than or equal to three lobes (80% vs 40%; odds ratio: 6.0; 95% confidence interval: 1.33-27.05; P = .02), and consolidation with accompanying ground-glass opacity (4% vs 33%; P = .031) were more common in SARS-CoV-2 group. Some clinical, laboratory, and radiological findings may help in the differential diagnosis of non-SARS-CoV-2 viruses from COVID-19. However, coinfections may occur, and a non-SARS-CoV-2 pathogen positivity does not exclude accompanying COVID-19., (© 2020 Wiley Periodicals LLC.)

Background: Diabetes is a risk factor for infection with coronaviruses. This study describes the demographic, clinical data, and outcomes of critically ill patients with diabetes and Middle East Respiratory Syndrome (MERS)., Methods: This retrospective cohort study was conducted at 14 hospitals in Saudi Arabia (September 2012-January 2018). We compared the demographic characteristics, underlying medical conditions, presenting symptoms and signs, management and clinical course, and outcomes of critically ill patients with MERS who had diabetes compared to those with no diabetes. Multivariable logistic regression analysis was performed to determine if diabetes was an independent predictor of 90-day mortality., Results: Of the 350 critically ill patients with MERS, 171 (48.9%) had diabetes. Patients with diabetes were more likely to be older, and have comorbid conditions, compared to patients with no diabetes. They were more likely to present with respiratory failure requiring intubation, vasopressors, and corticosteroids. The median time to clearance of MERS-CoV RNA was similar (23 days (Q1, Q3: 17, 36) in patients with diabetes and 21.0 days (Q1, Q3: 10, 33) in patients with no diabetes). Mortality at 90 days was higher in patients with diabetes (78.9% versus 54.7%, p < 0.0001). Multivariable regression analysis showed that diabetes was an independent risk factor for 90-day mortality (odds ratio, 2.09; 95% confidence interval, 1.18-3.72)., Conclusions: Half of the critically ill patients with MERS have diabetes; which is associated with more severe disease. Diabetes is an independent predictor of mortality among critically patients with MERS.

Understanding the transmission mechanism of SARS-CoV-2 is a prerequisite to effective control measures. To investigate the potential modes of SARS-CoV-2 transmission, 21 COVID-19 patients from 12-47 days after symptom onset were recruited. We monitored the release of SARS-CoV-2 from the patients' exhaled breath and systematically investigated environmental contamination of air, public surfaces, personal necessities, and the drainage system. SARS-CoV-2 RNA was detected in 0 of 9 exhaled breath samples, 2 of 8 exhaled breath condensate samples, 1 of 12 bedside air samples, 4 of 132 samples from private surfaces, 0 of 70 samples from frequently touched public surfaces in isolation rooms, and 7 of 23 feces-related air/surface/water samples. The maximum viral RNA concentrations were 1857 copies/m 3 in the air, 38 copies/cm 2 in sampled surfaces and 3092 copies/mL in sewage/wastewater samples. Our results suggest that nosocomial transmission of SARS-CoV-2 can occur via multiple routes. However, the low detection frequency and limited quantity of viral RNA from the breath and environmental specimens may be related to the reduced viral load of the COVID-19 patients on later days after symptom onset. These findings suggest that the transmission dynamics of SARS-CoV-2 differ from those of SARS-CoV in healthcare settings., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Coronavirus disease 2019 (COVID-19) is a disease caused by a new strain of coronavirus named as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Globally, since the outbreak, more than seven million confirmed cases of COVID-19 have been reported. The rapid spread and increase in the number of new cases is due to person-to-person transmission. To further control its transmission, early laboratory diagnosis of both asymptomatic and symptomatic patients is crucial. Presently, the COVID-19 diagnosis of infected individuals is dependent on computed tomography scanning and real-time polymerase chain reaction (PCR). The latter is considered more sensitive and efficient for early diagnosis. In this review, general comparisons are made (cases, fatality rate, incubation period, clinical features, and reservoirs) and diagnostic laboratory procedures (specimens, extraction methods, and positive rates by real-time PCR) are compared between SARS, Middle East Respiratory Syndrome, and SARS-2. In total, 8982 SARS-2 suspected patients specimen data were retrieved, in which 40.9% (n = 3678) were detected as positive by real-time PCR. The specimen-wise high detection rate was observed from bronchoalveolar lavage, followed by saliva, nasal swabs, and sputum. As the COVID-19 cases are persistently increasing, the selection of appropriate specimens and laboratory assay would help in rapid and timely diagnosis.

We compared SARS-CoV-2 detection rate of different respiratory specimens (nasopharyngeal swab [NPS], n=92; oropharyngeal swab [OPS], n=18; sputum, n=11). We also compared cycle threshold (Ct) values of paired specimen types obtained from the same patient on the same day. Then we characterized viral load kinetics of NPS (n=142), OPS (n=126), and sputum (n=75), during disease course. Sputum samples showed higher detection rate than NPS, and OPS exhibited the lowest detection rate. The median Ct values in NPS were significantly lower than in paired OPS, and higher than in paired sputum, respectively ( P <0.05). During the disease course, viral load was the lowest in OPS and the highest in sputum samples., (Copyright © 2021 by the Association of Clinical Scientists, Inc.)