Introduction of graphene oxide-supported multilayer-quantum dots nanofilm into multiplex lateral flow immunoassay: A rapid and ultrasensitive point-of-care testing technique for multiple respiratory viruses.

A lateral flow immunoassay (LFA) biosensor that allows the sensitive and accurate identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other common respiratory viruses remains highly desired in the face of the coronavirus disease 2019 pandemic. Here, we propose a multiplex LFA method for the on-site, rapid, and highly sensitive screening of multiple respiratory viruses, using a multilayered film-like fluorescent tag as the performance enhancement and signal amplification tool. This film-like three-dimensional (3D) tag was prepared through the layer-by-layer assembly of highly photostable CdSe@ZnS-COOH quantum dots (QDs) onto the surfaces of monolayer graphene oxide nanosheets, which can provide larger reaction interfaces and specific active surface areas, higher QD loads, and better luminescence and dispersibility than traditional spherical fluorescent microspheres for LFA applications. The constructed fluorescent LFA biosensor can simultaneously and sensitively quantify SARS-CoV-2, influenza A virus, and human adenovirus with low detection limits (8 pg/mL, 488 copies/mL, and 471 copies/mL), short assay time (15 min), good reproducibility, and high accuracy. Moreover, our proposed assay has great potential for the early diagnosis of respiratory virus infections given its robustness when validated in real saliva samples. Electronic Supplementary Material: Supplementary material (Section S1 Experimental section, Section S2 Calculation of the maximum number of QDs on the GO@TQD nanofilm, Section S3 Optimization of the LFA method, and Figs. S1-S17 mentioned in the main text) is available in the online version of this article at 10.1007/s12274-022-5043-6.

Clinical Distribution and Drug Resistance of Pseudomonas aeruginosa in Guangzhou, China from 2017 to 2021.

The aim of the current study was to analyse the distribution of antimicrobial drug resistance (AMR) among Pseudomonas aeruginosa (P. aeruginosa, PA) isolates from Guangdong Provincial People's Hospital (GDPH) from 2017 to 2021, and the impact of the COVID-19 outbreak on changes in the clinical distribution and drug resistance rate of P. aeruginosa to establish guidelines for empiric therapy. Electronic clinical data registry records from 2017 to 2021 were retrospectively analysed to study the AMR among P. aeruginosa strains from GDPH. The strains were identified by VITEK 2 Compact and MALDI-TOF MS, MIC method or Kirby-Bauer method for antibiotic susceptibility testing. The results were interpreted according to the CLSI 2020 standard, and the data were analysed using WHONET 5.6 and SPSS 23.0 software. A total of 3036 P. aeruginosa strains were detected in the hospital from 2017 to 2021, and they were primarily distributed in the ICU (n = 1207, 39.8%). The most frequent specimens were respiratory tract samples (59.6%). The detection rate for P. aeruginosa in 5 years was highest in September, and the population distribution was primarily male(68.2%). For the trend in the drug resistance rate, the 5-year drug resistance rate of imipenem (22.4%), aztreonam (21.5%) and meropenem (19.3%) remained at high levels. The resistance rate of cefepime decreased from 9.4% to 4.8%, showing a decreasing trend year by year (p < 0.001). The antibiotics with low resistance rates were aminoglycoside antibiotics, which were gentamicin (4.4%), tobramycin (4.3%), and amikacin (1.4%), but amikacin showed an increasing trend year by year (p = 0.008). Our analysis indicated that the detection rate of clinically resistant P. aeruginosa strains showed an upwards trend, and the number of multidrug-resistant (MDR) strains increased year by year, which will lead to stronger pathogenicity and mortality. However, after the outbreak of COVID-19 in 2020, the growth trend in the number of MDR bacteria slowed, presumably due to the strict epidemic prevention and control measures in China. This observation suggests that we should reasonably use antibiotics and treatment programs in the prevention and control of P. aeruginosa infection. Additionally, health prevention and control after the outbreak of the COVID-19 epidemic (such as wearing masks, washing hands with disinfectant, etc., which reduced the prevalence of drug resistance) led to a slowdown in the growth of the drug resistance rate of P. aeruginosa in hospitals, effectively reducing the occurrence and development of drug resistance, and saving patient's treatment costs and time.

Introduction of graphene oxide-supported multilayer-quantum dots nanofilm into multiplex lateral flow immunoassay: A rapid and ultrasensitive point-of-care testing technique for multiple respiratory viruses

A lateral flow immunoassay (LFA) biosensor that allows the sensitive and accurate identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other common respiratory viruses remains highly desired in the face of the coronavirus disease 2019 pandemic. Here, we propose a multiplex LFA method for the on-site, rapid, and highly sensitive screening of multiple respiratory viruses, using a multilayered film-like fluorescent tag as the performance enhancement and signal amplification tool. This film-like three-dimensional (3D) tag was prepared through the layer-by-layer assembly of highly photostable CdSe@ZnS−COOH quantum dots (QDs) onto the surfaces of monolayer graphene oxide nanosheets, which can provide larger reaction interfaces and specific active surface areas, higher QD loads, and better luminescence and dispersibility than traditional spherical fluorescent microspheres for LFA applications. The constructed fluorescent LFA biosensor can simultaneously and sensitively quantify SARS-CoV-2, influenza A virus, and human adenovirus with low detection limits (8 pg/mL, 488 copies/mL, and 471 copies/mL), short assay time (15 min), good reproducibility, and high accuracy. Moreover, our proposed assay has great potential for the early diagnosis of respiratory virus infections given its robustness when validated in real saliva samples. Electronic Supplementary Material Supplementary material (Section S1 Experimental section, Section S2 Calculation of the maximum number of QDs on the GO@TQD nanofilm, Section S3 Optimization of the LFA method, and Figs. S1–S17 mentioned in the main text) is available in the online version of this article at 10.1007/s12274-022-5043-6.

Pneumonia Caused by Coinfection with Cytomegalovirus and Pneumocystis jirovecii in an HIV-Negative Infant Diagnosed by Metagenomic Next-Generation Sequencing.

Background: Pneumonia produced by coinfection with Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) in infants and young children without timely diagnosis and treatment is often fatal due to the limitations of traditional tests. More accurate and rapid diagnostic methods for multiple infections are urgently needed. Case Presentation: Here, we report a case of a 2-month-old boy with pneumonia caused by Pneumocystis jirovecii (PJ) and cytomegalovirus (CMV) without HIV infection. Chest computed tomography (CT) showed massive exudative consolidation in both lungs. Microscopic examination of stained sputum and smear specimens and bacterial and fungal culture tests were all negative, and CMV nucleic acid and antibody tests were positive. After a period of antiviral and anti-infective therapy, pulmonary inflammation was not relieved. Subsequently, sputum and venous blood samples were analysed by metagenomic next-generation sequencing (mNGS), and the sequences of PJ and CMV were acquired. The patient was finally diagnosed with pneumonia caused by PJ and CMV coinfection. Anti-fungal combined with anti-viral therapy was given immediately. mNGS re-examination of bronchoalveolar lavage fluid (BALF) also revealed the same primary pathogen. Therapy was stopped due to the request of the patient's guardian. Hence, the child was discharged from the hospital and eventually died. Conclusion: This case emphasizes the combined use of mNGS and traditional tests in the clinical diagnosis of mixed lung infections in infants without HIV infection. mNGS is a new adjunctive diagnostic method that can rapidly discriminate multiple causes of pneumonia.

RT-LAMP assay for rapid detection of the R203M mutation in SARS-CoV-2 Delta variant.

The highly infectious Delta variant strain of SARS-CoV-2 remains globally dominant and undermines COVID-19 vaccines. Rapid detection of the Delta variant is crucial for the identification and quarantine of infected individuals. In this study, our aim was to design and validate a genotyping RT-LAMP method to detect Delta variants specifically. R203M in the N gene of SARS-CoV-2 was chosen as the Delta variant-specific mutation for genotyping. To target the R203M-harboring region and the conserved sequence of the N gene, two sets of primers were designed, and a Cq (quantification cycle) ratio-based RT-LAMP for SARS-CoV-2 and R203M detection was developed by analyzing the significant discrepancy in amplification efficiency of the two sets of primers. We validated the RT-LAMP method on 498 clinical specimens in parallel with RT-qPCR, and 84 Delta variants from 198 positive samples were determined by sequencing. Compared with traditional RT-qPCR analyses, RT-LAMP appears to be 100% accurate in detecting SARS-CoV-2 clinical samples. RT-LAMP has a good ability to distinguish between Delta and non-Delta variants under a Cq ratio threshold of 1.80. Furthermore, the AUC (area under the curve) of this method was 1.00; the sensitivity, specificity and accuracy were all 100%. In summary, we have proposed a rapid, accurate and cost-effective RT-LAMP method to detect SARS-CoV-2 and Delta variants, which may facilitate the surveillance of COVID-19.

Immune Response, Viral Shedding Time, and Clinical Characterization in COVID-19 Patients With Gastrointestinal Symptoms.

Background and Aims: Gastrointestinal (GI) symptoms are frequently observed in coronavirus disease (COVID-19) symptoms. Previous studies have mainly focused on epidemiology and characteristics in patients with GI symptoms, little is known about the roles of the immune response in susceptibility to and severity of infection. Here, we analyzed COVID-19 cases to determine immune response and clinical characteristics in COVID-19 patients with GI symptoms. Methods: Based on the presence of GI symptoms, 79 patients in Xuzhou were divided into GI and non-GI groups. A retrospective study investigating the clinical characteristics, selected laboratory abnormalities, immune response, treatment, and clinical outcome was performed to compare patients with or without GI symptoms. Results: Approximately 25% of patients reported at least one GI symptom. Our results showed significantly higher rates of fatigue, increased LDH, increased CK, higher percentage increase neutrophil-to-lymphocyte ratio (NLR), lymphopenia, and bilateral pneumonia in patients with GI symptoms. No significant changes in serum amylase (SAA), immunoglobulin (Ig) G, IgM, C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), viral shedding time, liver injury, and kidney injury between the two groups were observed. The clinical type on admission of patients with GI symptoms reported significantly higher rates of critical disease type (20 vs. 3.3%; p = 0.033). However, the survival rate did not differ between the two groups. Conclusions: Increase in total lymphocytes and NLR as well as the elevation of CRP, SAA, PCT, IL-6, CK, and LDH were closely associated with COVID-19 with GI symptoms, implying reliable indicators COVID-19 patients with GI symptoms were more likely to develop into a severe disease.

Clinical findings in a group of COVID-19 patients: a single-center retrospective study.

BACKGROUND: The coronavirus disease 2019 (COVID-19) is spreading rapidly across countries and has infected tens of millions of people all over the world. So far, the pandemic is ongoing globally, and the situation is still worsening. METHODS: In this retrospective, single-center cohort analysis, we included 25 adult inpatients with laboratory confirmed COVID-19 disease from the affiliated hospital of Xuzhou Medical University (Xuzhou, China). Epidemiological characterizations, clinical findings, and medical treatments were all reported. In addition, laboratory markers were investigated in terms of course of treatment. RESULTS: Epidemiological features and clinical findings were present for all 25 patients. Laboratory markers were identified due to temporal changes. After medical treatment, all patients were discharged home and recovering from the infection. CONCLUSIONS: This study provides a comprehensive overview of patients with COVID-19 disease in a single hospital. Some of the laboratory markers were statistically different during the course of the disease, which might serve as indicators in identifying patients with COVID-19 disease at an early stage of the infection.

Sensitive and Simultaneous Detection of SARS-CoV-2-Specific IgM/IgG Using Lateral Flow Immunoassay Based on Dual-Mode Quantum Dot Nanobeads.

A rapid and accurate method for detection of virus (SARS-CoV-2)-specific antibodies is important to contain the 2019 coronavirus disease (COVID-19) outbreak, which is still urgently needed. Here, we develop a colorimetric-fluorescent dual-mode lateral flow immunoassay (LFIA) biosensor for rapid, sensitive, and simultaneous detection of SARS-CoV-2-specific IgM and IgG in human serum using spike (S) protein-conjugated SiO2@Au@QD nanobeads (NBs) as labels. The assay only needs 1 µL of the serum sample, can be completed within 15 min, and is 100 times more sensitive than the colloidal gold-based LFIA. Two detection modes of our biosensor are available: the colorimetric mode for rapid screening of the patients with suspected SARS-CoV-2 infection without any special instrument and the fluorescent mode for sensitive and quantitative analyses to determine the concentrations of specific IgM/IgG in human serum and detect the infection early and precisely. We validated the proposed method using 16 positive serum samples from patients with COVID-19 and 41 negative samples from patients with other viral respiratory infections. The results demonstrated that combined detection of virus-specific IgM and IgG via SiO2@Au@QD LFIA can identify 100% of patients with SARS-CoV-2 infection with 100% specificity.

Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19.

The recent outbreak of human infections caused by SARS-CoV-2, the third zoonotic coronavirus has raised great public health concern globally. Rapid and accurate diagnosis of this novel pathogen posts great challenges not only clinically but also technologically. Metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methodologies. However, each has their own limitations. In this study, we developed an isothermal, CRISPR-based diagnostic for COVID-19 with near single-copy sensitivity. The diagnostic performances of all three technology platforms were also compared. Our study aimed to provide more insights into the molecular detection of SARS-CoV-2, and also to present a novel diagnostic option for this new emerging virus.

Laboratory diagnosis of coronavirus disease-2019 (COVID-19).

The outbreak of Coronavirus Disease-2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has threatened health worldwide. As of the end of 2020, there were nearly 10 million confirmed cases and nearly 5 million deaths associated with COVID-19. Rapid and early laboratory diagnosis of COVID-19 is the main focus of treatment and control. Molecular tests are the basis for confirmation of COVID-19, but serological tests for SARS-CoV-2 are widely available and play an increasingly important role in understanding the epidemiology of the virus and in identifying populations at higher risk for infection. Point-of-care tests have the advantage of rapid, accurate, portable, low cost and non-specific device requirements, which provide great help for disease diagnosis and detection. This review will discuss the performance of different laboratory diagnostic tests and platforms, as well as suitable clinical samples for testing, and related biosafety protection. This review shall guide for the diagnosis of COVID-19 caused by SARS-CoV-2.

Comparative analysis of laboratory indexes of severe and non-severe patients infected with COVID-19.

BACKGROUND: The pandemic coronavirus disease 2019 (COVID-19) has threaten the global health. The characteristics of laboratory findings of coronavirus are of great significance for clinical diagnosis and treatment. We found indicators that may most effectively predict a non-severe COVID-19 patient develop into a severe patient. METHODS: We conducted a meta-analysis to compare the laboratory findings of severe patients with non-severe patients with COVID-19 from searched articles. RESULTS: Through the analysis of laboratory examination information of patients with COVID-19 from 35 articles (5912 patients), we demonstrated that severe cases possessed higher levels of leukocyte (1.20-fold), neutrophil (1.33-fold), CRP (3.04-fold), PCT (2.00-fold), ESR (1.44-fold), AST (1.40-fold), ALT (1.34-fold), LDH (1.54-fold), CK (1.44-fold), CK-MB (1.39-fold), total bilirubin (1.14-fold), urea (1.28-fold), creatine (1.09-fold), PT (1.03-fold) and D-dimer (2.74-fold), as well as lower levels of lymphocytes (1.44-fold), eosinophil (2.00-fold), monocyte (1.08-fold), Hemoglobin (1.53-fold), PLT (1.15-fold), albumin (1.15-fold), and APTT (1.02-fold). Lymphocyte subsets and series of inflammatory cytokines were also different in severe cases with the non-severe ones, including lower levels of CD4 T cells (2.10-fold) and CD8 T cells (2.00-fold), higher levels of IL-1ß (1.02-fold), IL-6 (1.93-fold) and IL-10 (1.55-fold). CONCLUSIONS: Some certain laboratory inspections could predict the progress of the COVID-19 changes, especially lymphocytes, CRP, PCT, ALT, AST, LDH, D-dimer, CD4 T cells and IL6, which provide valuable signals for preventing the deterioration of the disease.

Different longitudinal patterns of nucleic acid and serology testing results based on disease severity of COVID-19 patients.

Effective strategy to mitigate the ongoing pandemic of 2019 novel coronavirus (COVID-19) require a comprehensive understanding of humoral responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the emerging virus causing COVID-19. The dynamic profile of viral replication and shedding along with viral antigen specific antibody responses among COVID-19 patients started to be reported but there is no consensus on their patterns. Here, we conducted a serial investigation on 21 individuals infected with SARS-CoV-2 in two medical centres from Jiangsu Province, including 11 non-severe COVID-19 patients, and 5 severe COVID-19 patients and 5 asymptomatic carriers based on nucleic acid test and clinical symptoms. The longitudinal swab samples and sera were collected from these people for viral RNA testing and antibody responses, respectively. Our data revealed different pattern of seroconversion among these groups. All 11 non-severe COVID-19 patients and 5 severe COVID-19 patients were seroconverted during hospitalization or follow-up period, suggesting that serological testing is a complementary assay to nucleic acid test for those symptomatic COVID-19 patients. Of note, immediate antibody responses were identified among severe cases, compared to non-severe cases. On the other hand, only one were seroconverted for asymptomatic carriers. The SARS-CoV-2 specific antibody responses were well-maintained during the observation period. Such information is of immediate relevance and would assist COVID-19 clinical diagnosis, prognosis and vaccine design.

Asymptomatic and Human-to-Human Transmission of SARS-CoV-2 in a 2-Family Cluster, Xuzhou, China.

We report epidemiologic, laboratory, and clinical findings for 7 patients with 2019 novel coronavirus disease in a 2-family cluster. Our study confirms asymptomatic and human-to-human transmission through close contacts in familial and hospital settings. These findings might also serve as a practical reference for clinical diagnosis and medical treatment.