A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application.

Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific sdaA gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the sdaA gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis mycobacterium (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions.

Computed tomography–based COVID–19 triage through a deep neural network using mask–weighted global average pooling

Background There is an urgent need to find an effective and accurate method for triaging coronavirus disease 2019 (COVID-19) patients from millions or billions of people. Therefore, this study aimed to develop a novel deep-learning approach for COVID-19 triage based on chest computed tomography (CT) images, including normal, pneumonia, and COVID-19 cases. Methods A total of 2,809 chest CT scans (1,105 COVID-19, 854 normal, and 850 non-3COVID-19 pneumonia cases) were acquired for this study and classified into the training set (n = 2,329) and test set (n = 480). A U-net-based convolutional neural network was used for lung segmentation, and a mask-weighted global average pooling (GAP) method was proposed for the deep neural network to improve the performance of COVID-19 classification between COVID-19 and normal or common pneumonia cases. Results The results for lung segmentation reached a dice value of 96.5% on 30 independent CT scans. The performance of the mask-weighted GAP method achieved the COVID-19 triage with a sensitivity of 96.5% and specificity of 87.8% using the testing dataset. The mask-weighted GAP method demonstrated 0.9% and 2% improvements in sensitivity and specificity, respectively, compared with the normal GAP. In addition, fusion images between the CT images and the highlighted area from the deep learning model using the Grad-CAM method, indicating the lesion region detected using the deep learning method, were drawn and could also be confirmed by radiologists. Conclusions This study proposed a mask-weighted GAP-based deep learning method and obtained promising results for COVID-19 triage based on chest CT images. Furthermore, it can be considered a convenient tool to assist doctors in diagnosing COVID-19.

Computed tomography-based COVID-19 triage through a deep neural network using mask-weighted global average pooling.

Background: There is an urgent need to find an effective and accurate method for triaging coronavirus disease 2019 (COVID-19) patients from millions or billions of people. Therefore, this study aimed to develop a novel deep-learning approach for COVID-19 triage based on chest computed tomography (CT) images, including normal, pneumonia, and COVID-19 cases. Methods: A total of 2,809 chest CT scans (1,105 COVID-19, 854 normal, and 850 non-3COVID-19 pneumonia cases) were acquired for this study and classified into the training set (n = 2,329) and test set (n = 480). A U-net-based convolutional neural network was used for lung segmentation, and a mask-weighted global average pooling (GAP) method was proposed for the deep neural network to improve the performance of COVID-19 classification between COVID-19 and normal or common pneumonia cases. Results: The results for lung segmentation reached a dice value of 96.5% on 30 independent CT scans. The performance of the mask-weighted GAP method achieved the COVID-19 triage with a sensitivity of 96.5% and specificity of 87.8% using the testing dataset. The mask-weighted GAP method demonstrated 0.9% and 2% improvements in sensitivity and specificity, respectively, compared with the normal GAP. In addition, fusion images between the CT images and the highlighted area from the deep learning model using the Grad-CAM method, indicating the lesion region detected using the deep learning method, were drawn and could also be confirmed by radiologists. Conclusions: This study proposed a mask-weighted GAP-based deep learning method and obtained promising results for COVID-19 triage based on chest CT images. Furthermore, it can be considered a convenient tool to assist doctors in diagnosing COVID-19.

Asymptomatic COVID-19 Patients Can Contaminate Their Surroundings: an Environment Sampling Study.

The contamination of patients' surroundings by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains understudied. We sampled the surroundings and the air of six negative-pressure non-intensive care unit (non-ICU) rooms in a designated isolation ward in Chengdu, China, that were occupied by 13 laboratory-confirmed coronavirus disease 2019 (COVID-19) patients who had returned from overseas travel, including 2 asymptomatic patients. A total of 44 of 112 (39.3%) surface samples were positive for SARS-CoV-2 as detected by real-time PCR, suggesting extensive contamination, although all of the air samples were negative. In particular, in a single room occupied by an asymptomatic patient, four sites were SARS-CoV-2 positive, highlighting that asymptomatic COVID-19 patients do contaminate their surroundings and impose risks for others with close contact. Placement of COVID-19 patients in rooms with negative pressure may bring a false feeling of safety, and the importance of rigorous environment cleaning should be emphasized.IMPORTANCE Although it has been well recognized that the virus SARS-CoV-2, the causative agent of COVID-19, can be acquired by exposure to fomites, surprisingly, the contamination of patients' surroundings by SARS-CoV-2 is largely unknown, as there have been few studies. We performed an environmental sampling study for 13 laboratory-confirmed COVID-19 patients and found extensive contamination of patients' surroundings. In particular, we found that asymptomatic COVID-19 patients contaminated their surroundings and therefore imposed risks for other people. Environment cleaning should be emphasized in negative-pressure rooms. The findings may be useful to guide infection control practice to protect health care workers.

Selective CB2 Receptor Agonist, HU-308, Reduces Systemic Inflammation in Endotoxin Model of Pneumonia-Induced Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) and sepsis are risk factors contributing to mortality in patients with pneumonia. In ARDS, also termed acute lung injury (ALI), pulmonary immune responses lead to excessive pro-inflammatory cytokine release and aberrant alveolar neutrophil infiltration. Systemic spread of cytokines is associated with systemic complications including sepsis, multi-organ failure, and death. Thus, dampening pro-inflammatory cytokine release is a viable strategy to improve outcome. Activation of cannabinoid type II receptor (CB2) has been shown to reduce cytokine release in various in vivo and in vitro studies. Herein, we investigated the effect of HU-308, a specific CB2 agonist, on systemic and pulmonary inflammation in a model of pneumonia-induced ALI. C57Bl/6 mice received intranasal endotoxin or saline, followed by intravenous HU-308, dexamethasone, or vehicle. ALI was scored by histology and plasma levels of select inflammatory mediators were assessed by Luminex assay. Intravital microscopy (IVM) was performed to assess leukocyte adhesion and capillary perfusion in intestinal and pulmonary microcirculation. HU-308 and dexamethasone attenuated LPS-induced cytokine release and intestinal microcirculatory impairment. HU-308 modestly reduced ALI score, while dexamethasone abolished it. These results suggest administration of HU-308 can reduce systemic inflammation without suppressing pulmonary immune response in pneumonia-induced ALI and systemic inflammation.

Computed tomography findings and clinical manifestations in different clinical types of coronavirus disease 2019

OBJECTIVE: Since the coronavirus disease 2019 (COVID-19) outbreak in Wuhan in 2019, the virus has spread rapidly. We investigated the clinical and computed tomography (CT) characteristics of different clinical types of COVID-19. MATERIALS AND METHODS: We retrospectively analyzed clinical and chest CT findings of 89 reverse transcription polymerase chain reaction confirmed cases from five medical centers in China. All the patients were classified into the common (n = 65), severe (n = 18), or fatal (n = 6) type. CT features included lesion distribution, location, size, shape, edge, density, and the ratio of lung lesions to extra-pulmonary lesions. A COVID-19 chest CT analysis tool (uAI-discover-COVID-19) was used to calculate the number of infections from the chest CT images. RESULTS: Fatal type COVID-19 is more common in older men, with a median age of 65 years. Fever was more common in the severe and fatal type COVID-19 patients than in the common type patients. Patients with fatal type COVID-19 were more likely to have underlying diseases. On CT examination, common type COVID-19 showed bilateral (68%), patchy (83%), ground-glass opacity (48%), or mixed (46%) lesions. Severe and fatal type COVID-19 showed bilateral multiple mixed density lesions (56%). The infection ratio (IR) increased in the common type (2.4 [4.3]), severe type (15.7 [14.3]), and fatal type (36.9 [14.2]). The IR in the inferior lobe of both lungs was statistically different from that of other lobes in common and severe type patients (P < 0.05). However, in the fatal type group, only the IR in the right inferior lung (RIL) was statistically different from that in the right superior lung(RUL), right middle lung (RML), and the left superior lung (LSL) (P < 0.05). CONCLUSION: The CT findings and clinical features of the various clinical types of COVID-19 pneumonia are different. Chest CT findings have unique characteristics in the different clinical types, which can facilitate an early diagnosis and evaluate the clinical course and severity of COVID-19.

Effects of growth hormone/estrogen/androgen on COVID-19-type proinflammatory responses in normal human lung epithelial BEAS-2B cells.

BACKGROUND: COVID-19 is a disease caused by SARS-CoV-2, which can cause mild to serious infections in humans. We aimed to explore the effect of growth hormone (GH)/estrogen/androgen in normal human lung epithelial BEAS-2B cells on COVID-19-type proinflammatory responses. METHODS: A BEAS-2B COVID-19-like proinflammatory cell model was constructed. After that, the cells were treated with GH, 17ß-estradiol (E2), and testosterone (Tes) for 24 h. CCK-8 assays were utilized to evaluate cell viability. The mRNA expression of ACE2, AGTR1, TMRRSS2, and ISG15 and the protein expression of ACE2, AGTR1, TMRRSS2, and ISG15 were measured by qRT‒PCR and Western blotting, respectively. ELISAs were performed to determine IL-6, MCP-1, MDA and SOD expression. Flow cytometry was used to measure ROS levels. Finally, MAPK/NF-κB pathway-related factor expression was evaluated. RESULTS: The COVID-19-type proinflammatory model was successfully constructed, and 1000 ng/mL RBD treatment for 24 h was selected as the condition for the model group for subsequent experiments. After RBD treatment, cell viability decreased, the mRNA expression of ACE2, AGTR1, TMRRSS2, and ISG15 and the protein expression of ACE2, AGTR1, TMRRSS2, and ISG15 increased, IL-6, MCP-1, MDA and ROS levels increased, and MDA levels decreased. The mRNA levels of MAPK14 and RELA increased, but the protein levels did not change significantly. In addition, phospho-MAPK14 and phospho-RELA protein levels were also increased. Among the tested molecules, E2 had the most pronounced effect, followed by GH, while Tes showed the opposite effect. CONCLUSION: GH/E2 alleviated inflammation in a COVID-19-type proinflammatory model, but Tes showed the opposite effect.

Dysregulation of Innate and Adaptive Immune Responses in Asymptomatic SARS-CoV-2 Infection with Delayed Viral Clearance.

Asymptomatic infection with SARS-CoV-2 is a major concern in the control of the COVID-19 pandemic. Many questions concerning asymptomatic infection remain to be answered, for example, what are the differences in infectivity and the immune response between asymptomatic and symptomatic infections? In this study, based on a cohort established by the Wuchang District Health Bureau of Wuhan in the early stage of the COVID-19 pandemic in Wuhan in 2019, we conducted a comprehensive analysis of the clinical, virological, immunological, and epidemiological data of asymptomatic infections. The major findings of this study included: 1) the asymptomatic cohort enrolled this study exhibited low-grade but recurrent activity of viral replication; 2) despite a lack of overt clinical symptoms, asymptomatic infections exhibited ongoing innate and adaptive immune responses; 3) however, the immune response from asymptomatic infections was not activated adequately, which may lead to delayed viral clearance. Given the fragile equilibrium between viral infection and host immunity, and the delayed viral clearance in asymptomatic individuals, close viral monitoring should be scheduled, and therapeutic intervention may be needed.

Anti-Inflammatory Effects of the Iron Chelator, DIBI, in Experimental Acute Lung Injury.

Iron plays a critical role in the immune response to inflammation and infection due to its role in the catalysis of reactive oxygen species (ROS) through the Haber-Weiss and Fenton reactions. However, ROS overproduction can be harmful and damage healthy cells. Therefore, iron chelation represents an innovative pharmacological approach to limit excess ROS formation and the related pro-inflammatory mediator cascades. The present study was designed to investigate the impact of the iron chelator, DIBI, in an experimental model of LPS-induced acute lung injury (ALI). DIBI was administered intraperitoneally in the early and later stages of lung inflammation as determined by histopathological evaluation. We found that lung tissues showed significant injury, as well as increased NF-κB p65 activation and significantly elevated levels of various inflammatory mediators (LIX, CXCL2, CCL5, CXCL10, IL-1𝛽, IL-6) 4 h post ALI induction by LPS. Mice treated with DIBI (80 mg/kg) in the early stages (0 to 2 h) after LPS administration demonstrated a significant reduction of the histopathological damage score, reduced levels of NF-κB p65 activation, and reduced levels of inflammatory mediators. Intravital microscopy of the pulmonary microcirculation also showed a reduced number of adhering leukocytes and improved capillary perfusion with DIBI administration. Our findings support the conclusion that the iron chelator, DIBI, has beneficial anti-inflammatory effects in experimental ALI.

The Effect and the Mechanism of Comprehensive Treatment on the Ocular Surface and the Visual Quality of Online Teachers with a Mild-to-Moderate Dry Eye Condition During the Early Phase of Coronavirus Disease 2019.

OBJECTIVE: To investigate the effect of comprehensive treatment on ocular surface function and the visual quality of online teachers with a mild-to-moderate dry eye condition during the early phase of coronavirus disease 2019 (COVID-19). METHODS: Secondary school online teachers diagnosed with a mild-to-moderate dry eye disease in our outpatient clinic from February to May 2020 were enrolled in this retrospective cross-section study, and all patients received dry eye comprehensive treatment. A questionnaire survey on eye-use habits, visual quality and dry eye-related indicators was collected before and after treatment (2 and 4 weeks). The changes and the correlations between indicators before and after treatment were compared. RESULTS: A total of 30 patients (15 females and 15 males) were included. After comprehensive treatment, patients had significantly higher central tear meniscus height (TMH), non-invasive first tear film breakup time (NIBUTf) and non-invasive average tear film breakup time (NIBUTav) than those before with statistical significance (P < 0.05). Lower ocular surface disease index (OSDI) and Meibomian gland scores were observed after treatment with statistical significance (P < 0.05). Objective scatter index (OSI), modulation transfer function (MTF) cutoff, strehl ratio (SR), and tear film objective scatter index (TF-OSI) were significantly improved after treatment (P < 0.05). Besides, TF-OSI was positively correlated with the changes in OSDI, Meibomian gland score, eye-use duration and OSI with statistical significance (P < 0.05), while it was negatively correlated with NIBUTf, NIBUTav, the TMH of the central lower eyelid, SR, sleep duration, conjunctival congestion and the MTF cutoff (P < 0.05), respectively. No correlation between TF-OSI and ciliary congestion was found (P > 0.05). CONCLUSION: Comprehensive treatment could effectively improve the symptoms and visual quality of online teachers with a mild-to-moderate dry eye condition during the early stage of COVID-19 pandemic.

Lymphocyte subsets as a predictor of severity and prognosis in COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) had become a worldwide health threat. Early prediction of the severity of COVID-19 patients was important for reducing death rate and controlling this disease. METHODS AND MATERIALS: A total of 301 patients confirmed with COVID-19 in Wuhan from 8 February to 10 April 2020 were included. Clinical data were collected and analyzed. Diagnostic and prognostic utility of blood cell counts and lymphocyte subsets in COVID-19 patients were investigated. The receiver operator characteristic curve (ROC) was used in discriminating the mild and severe/critical cases. RESULTS: There were difference in blood cell counts and lymphocyte subsets among mild, severe and critical patients, which were also influenced by comorbidities and duration of disease. The area under the ROC of lymphocyte, CD3+ T cells, CD4+ T cells, and CD8+ T cells were 0.718, 0.721, 0.718, and 0.670, which were higher than that of other hematological parameters. The optimal threshold was 1205, 691, 402, and 177 per µl, respectively. Patients with higher counts of lymphocyte, CD3+ T cells, CD4+ T cells, or CD8+ T cells were correlated with shorter length of stay in hospital (p < 0.05). Multivariable Cox regression analysis showed disease severity, CD3+ T cells counts and time when the nucleic acid turned negative were independent risk factors for in-hospital death of COVID-19 patients (p < 0.05). CONCLUSION: Blood cell counts and lymphocyte subsets correlated with severity of COVID-19.

Role of eosinophils in the diagnosis and prognostic evaluation of COVID-19.

Severe acute respiratory syndrome coronavirus 2 is responsible for the coronavirus disease 2019 (COVID-19) epidemic, which has severely affected global public health security. However, the diagnosis and treatment of the disease need further exploration. Therefore, this retrospective analysis was conducted on multiple indicators of peripheral blood in patients with COVID-19 to determine the role of leukocytes, lymphocytes, and eosinophils in the diagnosis and prognostic evaluation of COVID-19. Baseline information and clinical records of 40 patients were collected, including demographic data, disease status, medication, and laboratory routine. The correlation between the inspection indicators and disease classification, as well as prognostic factors, was analyzed. Decreased eosinophils were detected in 33 out of 40 patients with COVID-19 on admission, while lymphocytes and eosinophils were inversely related to the severity of the disease, according to the Spearman's correlation coefficient. Thus, it could be deduced that eosinophils have better sensitivity for the diagnosis of COVID-19 and play a major role similar to lymphocytes in assessing the prognosis of patients.

Survival potential and infectiousness of SARS-CoV-2 in environment

Coronavirus disease 2019 (COVID-19) spreads rapidly and widely in the world, which is mainly transmitted through respiratory droplets and contact with contaminated media. In this study, SARS-CoV-2 was found to have a similar stability to severe acute respiratory syndrome coronavirus (SARS-CoV) by analyzing its survival time on different subject surfaces and main influencing factors in related research. SARS-CoV-2 can survive for several days at various subject surfaces or media at room temperature (stainless steel: 2 days, plastic: 3 days, glass: 4 days, etc.), and SARS-CoV-2 can persist for longer time at low temperature and low relative humidity, which has caused severe threat to public health and has posed severe challenges to the prevention and control of COVID-19. According to available data, SARS-CoV-2 has the characteristics of high infectiousness and high covertness, similar to influenza A virus. By understanding the survival potential and infectiousness of SARS-CoV-2 in environment, targeted disinfection and effective protection can be implemented to reduce the incidence of COVID-19.

Myoglobin and C-reactive protein are efficient and reliable early predictors of COVID-19 associated mortality.

Since the emergence of SARS-CoV-2, numerous studies have been attempting to determine biomarkers, which could rapidly and efficiently predict COVID-19 severity, however there is lack of consensus on a specific one. This retrospective cohort study is a comprehensive analysis of the initial symptoms, comorbidities and laboratory evaluation of patients, diagnosed with COVID-19 in Huoshenshan Hospital, Wuhan, from 4th February to 12th March, 2020. Based on the data collected from 63 severely ill patients from the onset of symptoms till the full recovery or demise, we found not only age (average 70) but also blood indicators as significant risk factors associated with multiple organ failure. The blood indices of all patients showed hepatic, renal, cardiac and hematopoietic dysfunction with imbalanced coagulatory biomarkers. We noticed that the levels of LDH (85%, P < .001), HBDH (76%, P < .001) and CRP (65%, P < .001) were significantly elevated in deceased patients, indicating hepatic impairment. Similarly, increased CK (15%, P = .002), Cre (37%, P = 0.102) and CysC (74%, P = 0.384) indicated renal damage. Cardiac injury was obvious from the significantly elevated level of Myoglobin (52%, P < .01), Troponin-I (65%, P = 0.273) and BNP (50%, P = .787). SARS-CoV-2 disturbs the hemolymphatic system as WBC# (73%, P = .002) and NEUT# (78%, P < .001) were significantly elevated in deceased patients. Likewise, the level of D-dimer (80%, P < .171), PT (87%, P = .031) and TT (57%, P = .053) was elevated, indicating coagulatory imbalances. We identified myoglobin and CRP as specific risk factors related to mortality and highly correlated to organ failure in COVID-19 disease.

Sustainability of SARS-CoV-2 Induced Humoral Immune Responses in COVID-19 Patients from Hospitalization to Convalescence Over Six Months.

Understanding the persistence of antibody in convalescent COVID-19 patients may help to answer the current major concerns such as the risk of reinfection, the protection period of vaccination and the possibility of building an active herd immunity. This retrospective cohort study included 172 COVID-19 patients who were hospitalized in Wuhan. A total of 404 serum samples were obtained over six months from hospitalization to convalescence. Antibodies in the specimens were quantitatively analyzed by the capture chemiluminescence immunoassays (CLIA). All patients were positive for the anti-SARS-CoV-2 IgM/IgG at the onset of COVID-19 symptoms, and the IgG antibody persisted in all the patients during the convalescence. However, only approximately 25% of patients can detect the IgM antibodies, IgM against N protein (N-IgM) and receptor binding domain of S protein (RBD-IgM) at the 27th week. The titers of IgM, N-IgM and RBD-IgM reduced to 16.7%, 17.6% and 15.2% of their peak values respectively. In contrast, the titers of IgG, N-IgG and RBD-IgG peaked at 4-5th week and reduced to 85.9%, 62.6% and 87.2% of their peak values respectively at the end of observation. Dynamic behavior of antibodies and their correlation in age, gender and severity groups were investigated. In general, the COVID-19 antibody was sustained at high levels for over six months in most of the convalescent patients. Only a few patients with antibody reducing to an undetectable level which needs further attention. The humoral immune response against SARS-CoV-2 infection in COVID-19 patients exhibits a typical dynamic of acquired immunity.

Clinical characteristics of re-positive COVID-19 patients in Huangshi, China: A retrospective cohort study.

A cluster of patients with coronavirus disease 2019 (COVID-19) underwent repeated positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA tests after they were discharged from the hospital. We referred to them as re-positive (RP) patients in this study. We aimed to describe the clinical characteristics of these patients in a retrospective cohort study. After being treated for COVID-19, the patients underwent 14 days of quarantine following their discharge from the Huangshi Hospital of Traditional Chinese Medicine and the Huangshi Hospital of Youse. Two additional sequential SARS-CoV-2 RNA tests were performed at the end of quarantine. The median age of the 368 patients was 51 years, and 184 (50%) patients were female. A total of 23 RP patients were observed at follow-up. Using multivariate Cox regression analysis, risk factors associated with RP included a higher ratio of lymphocyte/white blood cell on admission (adjusted HR 7.038; 95% CI, 1.911-25.932; P = 0.0034), lower peak temperature during hospitalization (adjusted HR, 0.203; 95% CI, 0.093-0.443; P<0.0001), and the presence of comorbidities, particularly hypertension or chronic diseases in the respiratory system (adjusted HR, 3.883; 95% CI, 1.468-10.273; P = 0.0063). Antivirus treatment with arbidol was associated with a lower likelihood of re-positive outcomes (adjusted HR, 0.178; 95% CI, 0.045-0.709; P = 0.0144).