The current epidemic situation of coronavirus disease 2019 (COVID-19) still remained severe. As the National Clinical Research Center for Infectious Diseases, the First Affiliated Hospital of Zhejiang University School of Medicine is the primary medical care center for COVID-19 in Zhejiang province. Based on the present expert consensus carried out by National Health Commission and National Administration of Traditional Chinese Medicine, our team summarized and established an effective treatment strategy centered on "Four-Anti and Two-Balance" for clinical practice. The "Four-Anti and Two-Balance" strategy included antivirus, anti-shock, anti-hyoxemia, anti-secondary infection, and maintaining of water, electrolyte and acid base balance and microecological balance. Meanwhile, integrated multidisciplinary personalized treatment was recommended to improve therapeutic effect. The importance of early viralogical detection, dynamic monitoring of inflammatory indexes and chest radiograph was emphasized in clinical decision-making. Sputum was observed with the highest positive rate of RT-PCR results. Viral nucleic acids could be detected in 10%patients' blood samples at acute period and 50%of patients had positive RT-PCR results in their feces. We also isolated alive viral strains from feces, indicating potential infectiousness of feces.Dynamic cytokine detection was necessary to timely identifying cytokine storms and application of artificial liver blood purification system. The "Four-Anti and Two-Balance" strategy effectively increased cure rate and reduced mortality. Early antiviral treatment could alleviate disease severity and prevent illness progression, and we found lopinavir/ritonavir combined with abidol showed antiviral effects in COVID-19. Shock and hypoxemia were usually caused by cytokine storms. The artificial liver blood purification system could rapidly remove inflammatory mediators and block cytokine storm.Moreover, it also favored the balance of fluid, electrolyte and acid-base and thus improved treatment efficacy in critical illness. For cases of severe illness, early and also short period of moderate glucocorticoid was supported. Patients with oxygenation index below 200 mmHg should be transferred to intensive medical center. Conservative oxygen therapy was preferred and noninvasive ventilation was not recommended. Patients with mechanical ventilation should be strictly supervised with cluster ventilator-associated pneumonia prevention strategies. Antimicrobial prophylaxis was not recommended except for patients with long course of disease, repeated fever and elevated procalcitonin (PCT), meanwhile secondary fungal infection should be concerned.Some patients with COVID-19 showed intestinal microbial dysbiosis with decreased probiotics such as and , so nutritional and gastrointestinal function should be assessed for all patients.Nutritional support and application of prebiotics or probiotics were suggested to regulate the balance of intestinal microbiota and reduce the risk of secondary infection due to bacterial translocation. Anxiety and fear were common in patients with COVID-19. Therefore,we established dynamic assessment and warning for psychological crisis. We also integrated Chinese medicine in treatment to promote disease rehabilitation through classification methods of traditional Chinese medicine. We optimized nursing process for severe patients to promote their rehabilitation. It remained unclear about viral clearance pattern after the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Therefore, two weeks' quarantine for discharged patients was required and a regular following up was also needed.The Zhejiang experience and suggestions have been implemented in our center and achieved good results. However, since COVID-19 was a newly emerging disease, more work was warranted to improve strategies of prevention, diagnosis and treatment for COVID-19.
Betacoronavirus ; isolation & purification ; China ; epidemiology ; Coronavirus Infections ; diagnosis ; epidemiology ; therapy ; virology ; Disease Management ; Early Diagnosis ; Feces ; virology ; Humans ; Pandemics ; Pneumonia, Viral ; diagnosis ; epidemiology ; therapy ; virology ; Sputum ; virology

Bronchoalveolar Lavage Fluid/virology* ; COVID-19/virology* ; Feces/virology* ; Genome, Viral ; Humans ; Mouth Mucosa/virology* ; Multiplex Polymerase Chain Reaction ; Nasal Mucosa/virology* ; SARS-CoV-2/genetics* ; Sputum/virology* ; Whole Genome Sequencing

OBJECTIVEDevelopment and application of a real time fluorescent quantitative PCR (FQ-PCR) assay for detecting WU polyomavirus in children with low respiratory tract infections.

METHODSThe VP2 gene of WU polyomavirus was selected as the detection target, from which the real time primers and probes were designed. The standard curve was established by using recombinant plasmid as template. And the FQ-PCR assay for specific detection of WU polyomavirus was established. The specificity, sensitivity and reproducibility of the method were evaluated. Furthermore, the clinical specimens from children with respiratory tract infections collected in Wenling First People's Hospital were quantitatively detected using this method.

RESULTSIn this study, the FQ-PCR method was established to detect a specific fragment in VP2gene of WU polyomavirus. The standard curve coefficient R2 was 0.998. And this method can detect as low as 50 copies recombinant plasmid. The clinical specimens of sputum and throat swab from children with respiratory tract infections were quantitatively detected using this method. 7 sputum specimens were detected as WU polyomavirus positive in 700 sputum specimens, the positive ratio was 1.00%. No positive specimens were detected in 146 specimens of throat swabs and 846 blood samples from same patient population.

CONCLUSIONThe results indicated that the FQ-PCR assay method established in this study was specific, rapid and sensitive for detecting WU polyomavirus in children with lower respiratory tract infections. The sputum specimen is more suitable to be used for gene detection of WU polyomavirus than throat swab or blood.

Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Polyomavirus ; isolation & purification ; Real-Time Polymerase Chain Reaction ; methods ; Respiratory Tract Infections ; virology ; Sputum ; virology

WU polyomavirus, which was firstly discovered in 2007, is a new human polyomavirus belonging to Polyomaviridae and containing circular double-stranded genomic DNA. In this study, the 278 clinical sputum specimens from children under 5 years old were collected from Wenzhou Medical College affiliated Wenling First Hospital, Zhejiang Province. Based on identification assay of WU polyomavirus previously reported, a WU polyomavirus was identified from clinical samples successfully, the positive rate was 0.4%. The sequences of PCR products were identical to that of VP2 gene and large T antigen gene derived from WU polyomavirus reported. The above results strongly suggested that the WU polyomavirus isolated was firstly found in Chinese children with acute lower respiratory tract infections. This study provides a firm basis for further research of WU polyomavirus.
Base Sequence ; Child, Preschool ; Humans ; Infant ; Infant, Newborn ; Molecular Sequence Data ; Polymerase Chain Reaction ; Polyomavirus ; genetics ; isolation & purification ; Sputum ; virology

OBJECTIVETo examine the RNA of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in the blood and excretion of convalescent patient with SARS for prevention and treatment of the disease.

METHODSA total of 276 samples, including plasma, urine, feces and sputum, obtained from 23 convalescent patients with SARS were studied at 3 time-points at least 21 days after the onset of symptoms. RNA was extracted and nested reverse transcription-polymerase chain reaction (RT-PCR) was carried out using SARS-CoV specific primers.

RESULTSAmong the 276 samples, SARS-CoV RNA was detected in 6 cases (38.8%) by nested RT-PCR. The positive rates of SARS-CoV RNA was 5.8% in feces and 2.9% in sputum samples but SARS-CoV RNA was not detectable in plasma and urine of all the cases.

CONCLUSIONThe existence of SARS-CoV RNA in the excretion of some convalescent patients with SARS showed that the excretion from these patients should be carefully treated whilthe re-transmission of SARS by which, should be further studied.

Adolescent ; Adult ; Aged ; Convalescence ; Feces ; virology ; Female ; Humans ; Male ; Middle Aged ; RNA, Viral ; analysis ; blood ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; isolation & purification ; Severe Acute Respiratory Syndrome ; virology ; Sputum ; virology

BACKGROUND: It is crucial to understand the current status of clinical laboratory practices for the largest outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infections in the Republic of Korea to be well prepared for future emerging infectious diseases. METHODS: We conducted a survey of 49 clinical laboratories in medical institutions and referral medical laboratories. A short questionnaire to survey clinical laboratory practices relating to MERS-CoV diagnostic testing was sent by email to the directors and clinical pathologists in charge of the clinical laboratories performing MERS-CoV testing. The survey focused on testing volume, reporting of results, resources, and laboratory safety. RESULTS: A total of 40 clinical laboratories responded to the survey. A total of 27,009 MERS-CoV real-time reverse transcription PCR (rRT-PCR) tests were performed. Most of the specimens were sputum (73.5%). The median turnaround time (TAT) was 5.29 hr (first and third quartile, 4.11 and 7.48 hr) in 26 medical institutions. The median TAT of more than a half of the laboratories (57.7%) was less than 6 hr. Many laboratories were able to perform tests throughout the whole week. Laboratory biosafety preparedness included class II biosafety cabinets (100%); separated pre-PCR, PCR, and post-PCR rooms (88.6%); negative pressure pretreatment rooms (48.6%); and negative pressure sputum collection rooms (20.0%). CONCLUSIONS: Clinical laboratories were able to quickly expand their diagnostic capacity in response to the 2015 MERS-CoV outbreak. Our results show that clinical laboratories play an important role in the maintenance and enhancement of laboratory response in preparation for future emerging infections.
Clinical Laboratory Services/*standards ; Clinical Laboratory Techniques/instrumentation/methods ; Coronavirus Infections/*diagnosis/epidemiology/virology ; Disease Outbreaks ; Humans ; Middle East Respiratory Syndrome Coronavirus/genetics/isolation & purification ; RNA, Viral/analysis ; Real-Time Polymerase Chain Reaction ; Republic of Korea/epidemiology ; Sputum/virology ; Surveys and Questionnaires

OBJECTIVENucleic acid amplification (PCR) fluorogenic quantitative assay is used for the diagnosis of respiratory syncytial virus (RSV) infection. This study was designed to explore the sensitivity of PCR fluorogenic quantitative assay for ascertaining respiratory RSV infection and RSV infection conditions by detecting the presence of RSV-RNA related sequences in children.

METHODSBronchial and nasopharyngeal secretions specimens from 261 hospitalized children with respiratory tract infections from January 2007 to October 2008 were collected. Respiratory syncytial virus nucleic acid (RNA) in the specimens was measuredby PCR fluorogenic quantitative assay. Blood RSV-IgM was detected by enzyme linked immunosorbent assay (ELISA). The sensitivity for ascertaining respiratory RSV infection was compared between the two assays.

RESULTSThe RSV-RNA positive rate ascertained by PCR fluorogenic quantitative assay (38.7%) was significantly higher than blood RSV-IgM positive rate (21.1%) (p<0.01). The RSV-RNA positive rate (43.6%) in children at ages of less than 6 months was significantly higher than that in children at ages of 1 to three years (32.1%) (p<0.01). The RSV-RNA positive rate in children with bronchiolitis (58.5%) was the highest, followed by bronchopneumonia (38.2%) and acute bronchitis (20.0%).

CONCLUSIONSThe sensitivity of PCR fluorogenic quantitative assay for ascertaining respiratory RSV infection is higher. RSV is a major pathogen of lower respiratory tract infections in infants and young children. A higher rate of RSV infection is associated with a younger age. RSV infection is the most common in children with bronchiolitis.

Antibodies, Viral ; blood ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Fluorescence ; Humans ; Immunoglobulin M ; blood ; Infant ; Male ; Polymerase Chain Reaction ; methods ; RNA, Viral ; analysis ; Respiratory Syncytial Viruses ; genetics ; immunology ; isolation & purification ; Respiratory Tract Infections ; virology ; Sensitivity and Specificity ; Sputum ; virology

OBJECTIVES: An outbreak of acute febrile illness occurred in the Republic of Korea Air Force boot camp from May to July 2011. An epidemiological investigation of the causative agent, which was of a highly infective nature, was conducted. METHODS: Throat swabs were carried out and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay was performed to identify possible causative factors. RESULTS: The mean age of patients who had febrile illness during the study period was 20.24 years. The multiplex RT-PCR assay identified respiratory syncytial virus (RSV) as the causative agent. The main symptoms were sore throat (76.0%), sputum (72.8%), cough (72.1%), tonsillar hypertrophy (67.9%), and rhinorrhea (55.9%). The mean temperature was 38.75degreesC and the attack rate among the recruits was 15.7% (588 out of 3750 recruits), while the mean duration of fever was 2.3 days. The prognosis was generally favorable with supportive care but recurrent fever occurred in 10.1% of the patients within a month. CONCLUSIONS: This is the first epidemiological study of an RSV outbreak that developed in a healthy young adult group. In the event of an outbreak of an acute febrile illness of a highly infective nature in facilities used by a young adult group, RSV should be considered among the possible causative agents.
Adolescent ; Adult ; Antiviral Agents/therapeutic use ; Body Temperature ; Disease Outbreaks ; Humans ; Male ; Military Personnel ; Multiplex Polymerase Chain Reaction ; Oseltamivir/therapeutic use ; Pharynx/virology ; RNA, Viral/chemistry/genetics/metabolism ; Republic of Korea/epidemiology ; Respiratory Syncytial Virus Infections/drug therapy/*epidemiology/virology ; Respiratory Syncytial Viruses/*genetics/isolation & purification ; Sputum/virology ; Young Adult

BACKGROUND: Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. METHODS: We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). RESULTS: While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P<0.0001). CONCLUSIONS: The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.
Acetylcysteine/chemistry ; Citrates/chemistry ; Coronavirus Infections/diagnosis ; Deoxyribonuclease I/metabolism ; Endopeptidase K/metabolism ; Humans ; Middle East Respiratory Syndrome Coronavirus/genetics/*isolation & purification ; RNA, Viral/analysis/*isolation & purification/metabolism ; Real-Time Polymerase Chain Reaction ; Sputum/*virology

As of Apr. 22, 2020, the World Health Organization (2020) has reported over 2.4 million confirmed coronavirus disease 2019 (COVID-19) patients and 169 151 deaths. Recent articles have uncovered genomic characteristics and clinical features of COVID-19 (Chan et al., 2020; Chang et al., 2020; Guan et al., 2020; Zhu et al., 2020), while our understanding of COVID-19 is still limited. As suggested by guidelines promoted by the General Office of National Health Commission of the People's Republic of China (2020) (from Versions 1 to 6), discharged standards for COVID-19 were still dependent on viral real-time polymerase chain reaction (RT-PCR) tests of respiratory specimens, showing that recovered COVID-19 patients with twice negative RT-PCR could meet discharge criteria. Here, we examined two cases in which nucleic acid test results were inconsistent with clinical and radiological findings, leading to suboptimal care.
Adult ; Betacoronavirus ; China ; Clinical Laboratory Techniques ; Coronavirus Infections ; diagnosis ; transmission ; Female ; Humans ; Male ; Middle Aged ; Pandemics ; Patient Discharge ; Pneumonia, Viral ; diagnosis ; transmission ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Sputum ; virology