High-throughput nanopore targeted sequencing for efficient drug resistance assay of Mycobacterium tuberculosis.

Drug-resistant tuberculosis (TB), especially multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), is one of the urgent clinical problems and public health challenges. Culture-based phenotypic drug susceptibility testing (pDST) is time-consuming, and PCR-based assays are limited to hotspot mutations. In this study, we developed and validated a convenient and efficient approach based on high-throughput nanopore sequencing technology combined with multiplex PCR, namely nanopore targeted sequencing (NTS), to simultaneously sequence 18 genes associated with antibiotic resistance in Mycobacterium tuberculosis (MTB). The analytical performance of NTS was evaluated, and 99 clinical samples were collected to assess its clinical performance. The NTS results showed that MTB and its drug resistance were successfully identified in approximately 7.5 h. Furthermore, compared to the pDST and Xpert MTB/RIF assays, NTS provided much more drug resistance information, covering 14 anti-TB drugs, and it identified 20 clinical cases of drug-resistant MTB. The mutations underlying these drug-resistant cases were all verified using Sanger sequencing. Our approach for this TB drug resistance assay offers several advantages, including being culture-free, efficient, high-throughput, and highly accurate, which would be very helpful for clinical patient management and TB infection control.

Enhanced inflammation and suppressed adaptive immunity in COVID-19 with prolonged RNA shedding.

Little is known regarding why a subset of COVID-19 patients exhibited prolonged positivity of SARS-CoV-2 infection. Here, we found that patients with long viral RNA course (LC) exhibited prolonged high-level IgG antibodies and higher regulatory T (Treg) cell counts compared to those with short viral RNA course (SC) in terms of viral load. Longitudinal proteomics and metabolomics analyses of the patient sera uncovered that prolonged viral RNA shedding was associated with inhibition of the liver X receptor/retinoid X receptor (LXR/RXR) pathway, substantial suppression of diverse metabolites, activation of the complement system, suppressed cell migration, and enhanced viral replication. Furthermore, a ten-molecule learning model was established which could potentially predict viral RNA shedding period. In summary, this study uncovered enhanced inflammation and suppressed adaptive immunity in COVID-19 patients with prolonged viral RNA shedding, and proposed a multi-omic classifier for viral RNA shedding prediction.

Clinical features and viral RNA shedding of imported and local cases with COVID-19 in Wenzhou, China.

ABSTRACT: Wenzhou had the highest number of confirmed novel coronavirus 2019 (COVID-19) cases outside the Hubei province. The aim of this study was to identify the difference in clinical features and viral RNA shedding between the imported and local COVID-19 cases in Wenzhou.All patients with confirmed COVID-19 admitted to Wenzhou Sixth People's Hospital, Wenzhou Central Hospital Medical Group, from January 17 to February 11, 2020, were enrolled in this study. Data was analyzed and compared for the imported and local cases with regard to epidemiological, demographic, clinical, radiological features, and laboratory findings. Outcomes for the enrolled participants were followed up until May 7, 2020.Of the 136 cases, 50 were imported from Wuhan. The median age was 45 years and 73 (53.7%) were men. The most common symptoms at onset were fever (104 [76.5%]) and cough (85[62.5%]). Pleural effusion was more common among imported cases compared to local cases. The white blood cell count, neutrophil count, lymphocyte count and platelet count of the imported cases were significantly lower than those of the local cases, while the prothrombin time was significantly longer than that of the local cases. Severe and critically ill patients accounted for 15.4% and 2.9%, respectively. The median duration of SARS-CoV-2 RNA shedding from symptom onset was 26 days (IQR 17-32.3 days) and there were no significant differences in duration of viral RNA shedding between the two groups.The study findings suggest that imported cases from Wuhan were more likely to be severe compared to the local cases in Wenzhou. However, there was no difference between imported and local cases on the viral shedding among the COVID patients.

COVID-19 and early-stage lung cancer both featuring ground-glass opacities: a propensity score-matched study.

BACKGROUND: Radiological manifestations of coronavirus disease 2019 (COVID-19) featured ground-glass opacities (GGOs), especially in the early stage, which might create confusion in differential diagnosis with early lung cancer. We aimed to specify the radiological characteristics of COVID-19 and early lung cancer and to unveil the discrepancy between them. METHODS: One hundred and fifty-seven COVID-19 patients and 374 early lung cancer patients from four hospitals in China were retrospectively enrolled. Epidemiological, clinical, radiological, and pathological characteristics were compared between the two groups using propensity score-matched (PSM) analysis. RESULTS: COVID-19 patients had more distinct symptoms, tended to be younger (P<0.0001), male (P<0.0001), and had a higher body mass index (P=0.014). After 1:1 PSM, 121 matched pairs were identified. Regarding radiological characteristics, patients with a single lesion accounted for 17% in COVID-19 and 89% in lung cancer (P<0.0001). Most lesions were peripherally found in both groups. Lesions in COVID-19 involved more lobes (median 3.5 vs. 1; P<0.0001) and segments (median 6 vs. 1; P<0.0001) and tended to have multiple types (67%) with patchy form (54%). Early lung cancer was more likely to have a single type (92%) with oval form (66%). Also, COVID-19 and early lung cancer either had some distinctive features on computed tomography (CT) images. CONCLUSIONS: Both COVID-19 and early lung cancers showed GGOs, with similar but independent features. The imaging characteristics should be fully understood and combined with epidemiological history, pathogen detection, laboratory tests, short-term CT reexamination, and pathological results to aid differential diagnosis.

COVID-19 patients with progressive and non-progressive CT manifestations.

OBJECTIVE: To explore the clinical and radiological characteristics of COVID-19 patients with progressive and non-progressive CT manifestations. METHODS: 160 patients with COVID-19 were retrospectively included from Wenzhou and Wuhan, China. CT features including lesion position, attenuation, form and total scores (0-4) at the segment level were evaluated. Other images signs were also assessed. 65 patients were classified as progressive (group 1) and 95 as non-progressive CT (group 2) groups according to score changes between the initial and second CT. RESULTS: Symptoms onset-initial CT interval time in group 1 [5 (2, 7) days] were significantly shorter than that in group 2 [10 (8, 14) days] (P < 0.001). Group 2 had higher radiological scores, with more lobes and segments affected, and other CT signs (P < 0.05). In group 1, radiological scores, the number of lobes and segments affected as well as lesions in both peripheral and central distribution, mixed ground grass opacity and consolidation density, and patchy form increased in the second CT (P < 0.05). More reticular pattern, subpleural linear opacity and bronchial dilatation were also found (P < 0.05). CONCLUSION: Typically radiological characteristics of progressive CT patients could potentially help to predict changes and increase understanding of the natural history of COVID-19.

Gamma-Glutamyltransferase Elevation Is Frequent in Patients With COVID-19: A Clinical Epidemiologic Study.

A newly identified coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the infectious coronavirus disease 2019 (COVID-19), emerged in December 2019 in Wuhan, Hubei Province, China, and now poses a major threat to global public health. Previous studies have observed highly variable alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in patients with COVID-19. However, circulating levels of the cholangiocyte injury biomarker gamma-glutamyltransferase (GGT) have yet to be reported in the existing COVID-19 case studies. Herein, we describe the relationship between GGT levels and clinical and biochemical characteristics of patients with COVID-19. Our study is a retrospective case series of 98 consecutive hospitalized patients with confirmed COVID-19 at Wenzhou Central Hospital in Wenzhou, China, from January 17 to February 5, 2020. Clinical data were collected using a standardized case report form. Diagnosis of COVID-19 was assessed by symptomatology, reverse-transcription polymerase chain reaction (RT-PCR), and computed tomography scan. The medical records of patients were analyzed by the research team. Of the 98 patients evaluated, elevated GGT levels were observed in 32.7%; increased C-reactive protein (CRP) and elevated ALT and AST levels were observed in 22.5%, 13.3%, and 20.4%, respectively; and elevated alkaline phosphatase (ALP) and triglycerides (TGs) were found in 2% and 21.4%, respectively. Initially, in the 82 patients without chronic liver disease and alcohol history, age older than 40 years (P = 0.027); male sex (P = 0.0145); elevated CRP (P = 0.0366), ALT (P < 0.0001), and ALP (P = 0.0003); and increased TGs (P = 0.0002) were found to be associated with elevated GGT levels. Elevated GGT (P = 0.0086) and CRP (P = 0.0162) levels had a longer length of hospital stay. Conclusion: A sizable number of patients with COVID-19 infection have elevated serum GGT levels. This elevation supports involvement of the liver in persons with COVID-19.

Assessment of Hypokalemia and Clinical Characteristics in Patients With Coronavirus Disease 2019 in Wenzhou, China.

Importance: Severe acute respiratory syndrome coronavirus 2 has caused a global outbreak of coronavirus disease 2019 (COVID-19). Severe acute respiratory syndrome coronavirus 2 binds angiotensin-converting enzyme 2 of the rennin-angiotensin system, resulting in hypokalemia. Objective: To investigate the prevalence, causes, and clinical implications of hypokalemia, including its possible association with treatment outcomes, among patients with COVID-19. Design, Setting, and Participants: This cohort study was conducted at Wenzhou Central Hospital and Sixth People's Hospital of Wenzhou, Wenzhou, China, from January 11, 2020, to February 15, 2020. Participants included patients who received a diagnosis of COVID-19 according to the criteria issued by the Chinese Health Bureau and were admitted to the hospital. The patients were classified as having severe hypokalemia (plasma potassium <3 mmol/L), hypokalemia (plasma potassium 3-3.5 mmol/L), and normokalemia (plasma potassium >3.5 mmol/L). The clinical features, therapy, and outcomes were compared between the 3 groups. Data analysis was conducted in March 2020. Interventions: The patients were given general support and antiviral therapy. Their epidemiological and clinical features were collected. Main Outcomes and Measures: The prevalence of hypokalemia and response to treatment with potassium supplements were measured by analyzing plasma and urine potassium levels. Results: One hundred seventy-five patients (87 female patients [50%]; mean [SD] age, 45 [14] years) were classified as having severe hypokalemia (31 patients [18%]), hypokalemia (64 patients [37%]), and normokalemia (80 patients [46%]). Patients with severe hypokalemia had statistically significantly higher body temperature (mean [SD], 37.6 °C [0.9 °C]) than the patients with hypokalemia (mean [SD], 37.2 °C [0.7 °C]; difference, 0.4 °C; 95% CI, 0.2-0.6 °C; P = .02) and the patients with normokalemia (mean [SD], 37.1 °C [0.8 °C]; difference, 0.5 °C; 95% CI, 0.3-0.7 °C; P = .005). Patients with higher levels of hypokalemia also had higher creatine kinase levels (severe hypokalemia, mean [SD], 200 [257] U/L [median, 113 U/L; interquartile range {IQR}, 61-242 U/L]; hypokalemia, mean [SD], 97 [85] U/L; and normokalemia, mean [SD], 82 [57] U/L), higher creatine kinase-MB fraction (severe hypokalemia, mean [SD], 32 [39] U/L [median, 14 U/L; IQR, 11-36 U/L]; hypokalemia, mean [SD], 18 [15] U/L; and normokalemia, mean [SD], 15 [8] U/L), higher lactate dehydrogenase levels (mean [SD], severe hypokalemia, 256 [88] U/L; hypokalemia, 212 [59] U/L; and normokalemia, 199 [61] U/L), and higher C-reactive protein levels (severe hypokalemia, mean [SD], 29 [23] mg/L; hypokalemia, mean [SD], 18 [20] mg/L [median, 12, mg/L; IQR, 4-25 mg/L]; and normokalemia, mean [SD], 15 [18] mg/L [median, 6 U/L; IQR, 3-17 U/L]). Of 40 severely and critically ill patients, 34 (85%) had hypokalemia. Patients with severe hypokalemia were given potassium at a dose of 40 mEq per day, for a total mean (SD) of 453 (53) mEq potassium chloride, during the hospital stay. The patients responded well to potassium supplements as they recovered. Conclusions and Relevance: The correction of hypokalemia is challenging because of continuous renal potassium loss resulting from the degradation of angiotensin-converting enzyme 2. The high prevalence of hypokalemia among patients with COVID-19 suggests the presence of disordered rennin-angiotensin system activity, which increases as a result of reduced counteractivity of angiotensin-converting enzyme 2, which is bound by severe acute respiratory syndrome coronavirus 2.

Asiaticoside attenuates bleomycin-induced pulmonary fibrosis in A2aR-/- mice by promoting the BMP7/Smad1/5 signaling pathway.

Idiopathic Pulmonary fibrosis(PF)is a chronic progressive disease, which is a lack of effective treatment,and the pathogenesis of IPF is not fully elucidated. Asiaticoside(AS) is isolated from Centella asiatica and has the effect of promoting scar healing and reducing scar formation. However,its possible role in idiopathic pulmonary fibrosis remains unclear. Adenosine A2A receptor (A2AR) is reported a protective factor in pulmonary fibrosis, and the bone morphogenetic protein 7 (BMP7) signaling pathway plays a crucial role in fibrosis in multiple organs. But the impact of A2AR on the BMP7 pathway has not yet been reported. Therefore, we hypothesized AS may promote the expression of A2AR, and then influence the BMP7/Smad1/5 pathway to alleviate pulmonary fibrosis. A2AR-/- mice and wild-type (WT) mice were administered bleomycin (BLM) by intratracheal injection. AS (50 mg/kg/d) was given daily for 28 days. AS reduced collagen deposition in lung tissue, interstitial lung inflammation. Furthermore, AS promoted A2AR expression and BMP7 pathway. Collectively, AS may attenuate BLM-induced pulmonary fibrosis by upregulating the BMP7 signaling pathway through A2AR.

Quantitative computed tomography of the coronavirus disease 2019 (COVID-19) pneumonia.

OBJECTIVE: To quantify coronavirus diseases 2019 (COVID-19) pneumonia and to explore whether quantitative computer tomography (CT) could be used to assess severity on admission. MATERIALS AND METHODS: From January 17 to February 9, 2020, 38 hospitalized patients with COVID-19 pneumonia were consecutively enrolled in our hospitals. All clinical data and the chest CT on admission were retrospectively reviewed and analyzed. Firstly, a quantitative method based on multi-scale convolutional neural networks was used to assess the infected lung segments and this was compared with the semi-quantitative method. Secondly, the quantitative method was tested with laboratory results and the pneumonia severity index (PSI) by correlation analyses. Thirdly, both quantitative and semi-quantitative parameters between patients with different PSI were compared. RESULTS: Thirty cases were finally enrolled: 16 (53.33%) of them were male, and the mean age was 48 years old. The interval from onset symptoms to first chest CT scan was 8 days. The proportion of ground glass opacity (GGO), consolidation and the total lesion based on the quantitative method was positively correlated with the semi-quantitative CT score (P < 0.001 for all; rs = 0.88, 0.87, 0.90), CRP (P = 0.0278, 0.0168, 0.0078; rs = 0.40, 0.43, 0.48) and ESR (P = 0.0296, 0.0408, 0.0048; rs = 0.46, 0.44, 0.58), respectively, and was negatively correlated with the lymphocyte count (P = 0.0222, 0.0024, 0.0068; rs = -0.42, -0.53, -0.48). There was a positive correlation trend between the proportion of total infection and the pneumonia severity index (P = 0.0994; rs = 0.30) and a tendency that patients with severe COVID-19 pneumonia had higher percentage of consolidation and total infection (P = 0.0903, 0.0989). CONCLUSIONS: Quantitative CT may have potential in assessing the severity of COVID-19 pneumonia on admission.

Clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (COVID-19):A multi-center study in Wenzhou city, Zhejiang, China.

BACKGROUND: Little is known about COVID-19 outside Hubei. The aim of this paper was to describe the clinical characteristics and imaging manifestations of hospitalized patients with confirmed COVID-19 infection in Wenzhou, Zhejiang, China. METHODS: In this retrospective cohort study, 149 RT-PCR confirmed positive patients were consecutively enrolled from January 17th to February 10th, 2020 in three tertiary hospitals of Wenzhou. Outcomes were followed up until Feb 15th, 2020. FINDINGS: A total of 85 patients had Hubei travel/residence history, while another 49 had contact with people from Hubei and 15 had no traceable exposure history to Hubei. Fever, cough and expectoration were the most common symptoms, 14 patients had decreased oxygen saturation, 33 had leukopenia, 53 had lymphopenia, and 82 had elevated C-reactive protein. On chest computed tomography (CT), lung segments 6 and 10 were mostly involved. A total of 287 segments presented ground glass opacity, 637 presented mixed opacity and 170 presented consolidation. Lesions were more localized in the peripheral lung with a patchy form. No significant difference was found between patients with or without Hubei exposure history. Seventeen patients had normal CT on admission of these, 12 had negative findings even10 days later. INTERPRETATION: Most patients presented with a mild infection in our study. The imaging pattern of multifocal peripheral ground glass or mixed opacity with predominance in the lower lung is highly suspicious of COVID-19 in the first week of disease onset. Nevetheless, some patients can present with a normal chest finding despite testing positive for COVID-19. FUNDING: We did not receive any fundings.

Prevalence and types of drug-resistant variants in Chinese patients with acute hepatitis B.

The presence of therapy-associated hepatitis B virus (HBV) variants is the main drawback of antiviral therapy for HBV infection. Moreover, drug-resistant variants are more insensitive to a second agent and more therapy-associated mutations will be present. To apply better nucleos(t)ide analogues (NA) and reduce the occurrence of resistance, the prevalence and types of drug-resistant mutations in acute hepatitis B patients were investigated in this study. One hundred three HBV DNA-positive patients with symptomatic acute hepatitis B that were observed from 2011 to 2013 were enrolled. Direct polymerase chain reaction sequencing was used firstly to screen HBV reverse-transcriptase domain to detect HBV mutants. Five lamivudine-resistant variants were identified. Clonal sequencing was performed for 5 resistance-positive samples and 10 other random samples. Interestingly, all detected samples harbored drug-resistant mutations, although with different percentage. Thirteen harbored lamivudine-related alone (five) or together with other NA related mutations (five with adefovir, one with entecavir, and one with telbivudine), and two of them harbored adefovir-related mutations. Also, mutations associated with four currently used NA were all detected, and the frequency is in accordance with the popularity of NA used in clinical practice. These data suggest that drug-resistant variants are present in patients with acute hepatitis B and NA should be applied more carefully for chronic hepatitis B patients developed from acute hepatitis B.

[Genotyping of HBV DNA in Shenzhen and clinical manifestations].

OBJECTIVE: To detect HBV DNA and its genotypes. METHODS: The 6 isoforms of HBV DNA was detected out using of different probes by Polymerase Chain Reaction and Nucleic Acid hybridization. RESULTS: Of 150 HBV DNA positive patients who lived in Shenzhen, 50 samples (33%) are type B, 36 samples (24%) are type C, 13 samples (9%) are type D, 3 samples is type F, 1 sample is type A, 48 samples (31%) are mixed type. The ALT value was significantly higher in genotype B than in genotype C. HBe positivity were higher in genotype B than genotype C. HBeAg positivity were higher in genotype C than in genotype B. There are not obvious relations between genotype and age or sex. CONCLUSION: In the detected samples, the major genotype of HBV DNA is type B, several are type C, D. The type E haven't been found. There are some relations between all kinds of genotypes and the severity of hepatitis B.

[Expression of P27 protein and cyclin E in colon cancer].

BACKGROUND & OBJECTIVE: P27 protein and cyclin E were negative cell cycle regulators. Until the present, the influence of P27 protein and cyclin E on progression of colon cancer was unclear. The aim of this study was to observe the expression features of P27 protein and cyclin E in the tissues of colon neoplasms, and to investigate the relationship between colon neoplasms and tumor special growth factor (TSGF). METHODS: Sixty-nine cases of samples included 23 normal tissues, 28 colon polyps (13 inflammatory polyps and 15 adenomatous polyps), and 18 colon carcinomas. The location and expression of P27 protein and cyclin E were determined using immunohistochemical method in all samples. These samples were diagnosed using formal pathological techniques simultaneously; the relationship between colon neoplasms and TSGF was also investigated. RESULTS: The positive signal of P27 and cyclin E was found mainly in the cytoplasm and extracellular matrix of normal colon tissues, inflammatory polyps, and adenomatous polyps. Less amount of positive expression product of P27 protein and cyclin E was observed in colon carcinoma cells; and the positive signal was only located in the cytoplasm of gland-like cells. The content of TSGF in colon carcinoma tissues was significantly higher than that in normal tissues (117.3+/-57.02 versus 64.16+/-27.5,P< 0.01), but there was no significant difference between colon carcinoma tissues and inflammatory polyp tissues (117.3+/-57.02 versus 92.5+/-47.9,P >0.05). CONCLUSION: P27 protein and cyclin E participate in the adjustment process of colon neoplasm occurrence and progression. The reduced expression of P27 protein and cyclin E may indicate the possibility of colon carcinoma.