Objective: The aim of the present study was to evaluate the performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one dried blood spot (DBS) as an alternative sample to plasma. Method: A total of 571 paired DBS/plasma samples were collected from men who have sex with men (MSM) and injection drug users (IDUs), and serological and molecular assays were performed. Using plasma results as the reference standard, the performance of DBS tests for HIV-1 RNA, HIV-1 DNA, and HCV RNA was evaluated. Pearson's correlation coefficients and Bland-Altman analysis were performed to assess the correlation and concordance between DBS and plasma. Results: Among paired plasma/DBS samples with detectable HIV-1 RNA and HCV RNA, five samples (5/32) were not detectable in DBS, while measurable HIV-1 RNA levels were present in plasma (1.44 to 3.99 log Conclusion The performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one DBS was acceptable. DBS, as an alternative sample to plasma, may be a viable option for the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA in resource-limited settings or for individuals living in areas that are difficult to access.
DNA, Viral/analysis* ; Diagnostic Tests, Routine/methods* ; Dried Blood Spot Testing/methods* ; HIV Infections/diagnosis* ; HIV-1/isolation & purification* ; Hepacivirus/isolation & purification* ; Hepatitis C/diagnosis* ; RNA, Viral/analysis* ; Sensitivity and Specificity ; Specimen Handling/methods* ; Syphilis/diagnosis* ; Treponema pallidum/isolation & purification*

Antibodies, Neutralizing ; Antibodies, Viral/isolation & purification* ; B-Lymphocytes/virology* ; COVID-19/virology* ; Humans ; Immunity/genetics* ; RNA, Viral/immunology* ; SARS-CoV-2/pathogenicity* ; Single-Cell Analysis

We optimized a fluorescent quantitative polymerase chain reaction (qPCR) assay system for rapid and real time detection of SARS-CoV-2 RNA. The results show that the lowest dilution of RNA samples used for the detection of SARS-CoV-2 RNA could reach 1/10 000 (the initial value is set as 10 ng/μL). Moreover, the cycle threshold (Ct) for samples of clinically diagnosed COVID-19 was lower than 35 or 40. The sensitivity of this method was satisfactory. The results were consistent with those of the COVID-19 detection kit on the market under the same conditions, but the number of cycles required was shortened by about 2. Therefore, the optimized assay developed in this study can be used in screening and early clinical diagnosis. Our work provides a tool to facilitate rapid clinical diagnosis of COVID-19.
Betacoronavirus ; genetics ; isolation & purification ; Coronavirus Infections ; diagnosis ; virology ; Early Diagnosis ; Humans ; Pandemics ; Pneumonia, Viral ; diagnosis ; virology ; Polymerase Chain Reaction ; methods ; standards ; RNA, Viral ; analysis ; genetics ; Sensitivity and Specificity ; Time Factors

Acute Kidney Injury ; etiology ; Betacoronavirus ; Coronavirus Infections ; complications ; Humans ; Kidney ; enzymology ; Pandemics ; Peptidyl-Dipeptidase A ; physiology ; Pneumonia, Viral ; complications ; Sequence Analysis, RNA ; methods ; Serine Endopeptidases ; physiology ; Single-Cell Analysis ; methods

OBJECTIVE: To investigate the clinical outcome of patients with moderate type of coronavirus disease 2019 (COVID-19) after discharge by retesting viral nucleic acid. METHODS: Seven patients with moderate COVID-19 met the discharge criteria enacted by National Health Commission were quarantined in hospital for 7 days, then continuously quarantined at home for 4 weeks after discharged. During the quarantined period, the symptoms and signs were documented, and sputum or nasal swab and feces samples were collected to test SARS-CoV-2 nucleic acid by RT-PCR method. RESULTS: There was no symptoms and signs during the quarantine period in all 7 patients. However, respiratory swabs from 3 patients were confirmed positive of SARS-CoV-2 nucleic acid at 5 to 7 days after they met the discharge criteria. CONCLUSIONS There is a relatively high incidence of positive viral nucleic acid in patients met the discharge criteria, and it is suggested that patients met the current discharge criteria should be quarantined in hospital for another 7 days and the follow-up viral testing is necessary.
Betacoronavirus ; isolation & purification ; Coronavirus Infections ; diagnosis ; Feces ; chemistry ; virology ; Follow-Up Studies ; Humans ; Pandemics ; Patient Discharge ; statistics & numerical data ; Pneumonia, Viral ; diagnosis ; Quarantine ; statistics & numerical data ; RNA, Viral ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtype-specific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.
Adult ; Aged ; Aged, 80 and over ; Aging ; genetics ; immunology ; Betacoronavirus ; CD4-Positive T-Lymphocytes ; metabolism ; Cell Lineage ; Chromatin Assembly and Disassembly ; Coronavirus Infections ; immunology ; Cytokine Release Syndrome ; etiology ; immunology ; Cytokines ; biosynthesis ; genetics ; Disease Susceptibility ; Flow Cytometry ; methods ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Gene Rearrangement ; Humans ; Immune System ; cytology ; growth & development ; immunology ; Immunocompetence ; genetics ; Inflammation ; genetics ; immunology ; Mass Spectrometry ; methods ; Middle Aged ; Pandemics ; Pneumonia, Viral ; immunology ; Sequence Analysis, RNA ; Single-Cell Analysis ; Transcriptome ; Young Adult

INTRODUCTION: Pregnant women are reported to be at increased risk of severe coronavirus disease 2019 (COVID-19) due to underlying immunosuppression during pregnancy. However, the clinical course of COVID-19 in pregnancy and risk of vertical and horizontal transmission remain relatively unknown. We aim to describe and evaluate outcomes in pregnant women with COVID-19 in Singapore. METHODS: Prospective observational study of 16 pregnant patients admitted for COVID-19 to 4 tertiary hospitals in Singapore. Outcomes included severe disease, pregnancy loss, and vertical and horizontal transmission. RESULTS: Of the 16 patients, 37.5%, 43.8% and 18.7% were infected in the first, second and third trimesters, respectively. Two gravidas aged ≥35 years (12.5%) developed severe pneumonia; one patient (body mass index 32.9kg/m2) required transfer to intensive care. The median duration of acute infection was 19 days; one patient remained reverse transcription polymerase chain reaction (RT-PCR) positive >11 weeks from diagnosis. There were no maternal mortalities. Five pregnancies produced term live-births while 2 spontaneous miscarriages occurred at 11 and 23 weeks. RT-PCR of breast milk and maternal and neonatal samples taken at birth were negative; placenta and cord histology showed non-specific inflammation; and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immunoglobulins were elevated in paired maternal and umbilical cord blood (n=5). CONCLUSION The majority of COVID-19 infected pregnant women had mild disease and only 2 women with risk factors (obesity, older age) had severe infection; this represents a slightly higher incidence than observed in age-matched non-pregnant women. Among the women who delivered, there was no definitive evidence of mother-to-child transmission via breast milk or placenta.
Abortion, Spontaneous/epidemiology* ; Adult ; COVID-19/transmission* ; COVID-19 Nucleic Acid Testing ; COVID-19 Serological Testing ; Cohort Studies ; Disease Transmission, Infectious/statistics & numerical data* ; Female ; Fetal Blood/immunology* ; Humans ; Infectious Disease Transmission, Vertical/statistics & numerical data* ; Live Birth/epidemiology* ; Maternal Age ; Milk, Human/virology* ; Obesity, Maternal/epidemiology* ; Placenta/pathology* ; Pregnancy ; Pregnancy Complications, Infectious/physiopathology* ; Pregnancy Outcome/epidemiology* ; Pregnancy Trimester, First ; Pregnancy Trimester, Second ; Prospective Studies ; RNA, Viral/analysis* ; Risk Factors ; SARS-CoV-2 ; Severity of Illness Index ; Singapore/epidemiology* ; Umbilical Cord/pathology* ; Young Adult

Zika virus (ZIKV) is one of the pathogens which is transmitted world widely, but there are no effective drugs and vaccines. Whole genome sequencing (WGS) of viruses could be applied to viral pathogen characterization, diagnosis, molecular surveillance, and even finding novel pathogens. We established an improved method using direct RNA sequencing with Nanopore technology to obtain WGS of ZIKV, after adding poly (A) tails to viral RNA. This established method does not require specific primers, complimentary DNA (cDNA) synthesis, and polymerase chain reaction (PCR)-based enrichment, resulting in the reduction of biases as well as of the ability to find novel RNA viruses. Nanopore technology also allows to read long sequences. It makes WGS easier and faster with long-read assembly. In this study, we obtained WGS of two strains of ZIKV following the established protocol. The sequenced reads resulted in 99% and 100% genome coverage with 63.5X and 21,136X, for the ZIKV PRVABC59 and MR 766 strains, respectively. The sequence identities of the ZIKV PRVABC59 and MR 766 strains for each reference genomes were 98.76% and 99.72%, respectively. We also found that the maximum length of reads was 10,311 bp which is almost the whole genome size of ZIKV. These long-reads could make overall structure of whole genome easily, and WGS faster and easier. The protocol in this study could provide rapid and efficient WGS that could be applied to study the biology of RNA viruses including identification, characterization, and global surveillance.
Bias (Epidemiology) ; Biology ; Diagnosis ; DNA ; Genome ; Genome Size ; Methods ; Nanopores ; Polymerase Chain Reaction ; RNA Viruses ; RNA ; RNA, Viral ; Sequence Analysis, RNA ; Tail ; Vaccines ; Zika Virus

OBJECTIVE: Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis (TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease. METHODS: A reverse-transcription recombinase-aided amplification (RT-RAA) assay was developed. This assay can be completed in one closed tube at 39 °C within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type (WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay. RESULTS: The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units (pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay. CONCLUSION A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.
Encephalitis Viruses, Tick-Borne ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; RNA, Viral ; analysis

OBJECTIVE: To investigate the molecular epidemiological characteristics of norovirus (NoV) among children with acute gastroenteritis in Tianjin in 2017. METHODS: A total of 758 stool specimens were collected from the children with acute gastroenteritis possibly caused by viral infection in Tianjin Children's Hospital between January and December, 2017. Quantitative real-time RT-PCR was used for primary screening of NoV, and conventional RT-PCR was used for gene amplification, sequencing and genotype identification of the VP1 region of capsid protein in positive specimens. RESULTS: Among the 758 specimens, 241 (31.8%) were found to have GII NoV. Sequencing of the VP1 region of capsid protein in positive specimens showed that among the 241 specimens with GII NoV, 69 (28.6%) had GII.4 subtype, 51 (21.2%) had GII.3 subtype, 24 (10.0%) had GII.2 subtype, and 18 (7.5%) had other subtypes. There was a significant difference in NoV detection rate between different age groups (P=0.018), and the 1- <4 years group had the highest NoV detection rate (37.3%). There was also a significant difference in NoV detection rate across seasons (P<0.001), and there was a highest NoV detection rate in winter (48.1%). Twenty-seven children (3.6%) had co-infections with NoV and rotavirus. CONCLUSIONS NoV is one of the major pathogens of the children with acute gastroenteritis from Tianjin in 2017. GII genotype, especially GII.4 subtype, is the prevalent strain. NoV infection is commonly seen in children less than 4 years and reaches the peak in winter. Some children are found to have co-infections with rotavirus.
Caliciviridae Infections ; Child ; China ; epidemiology ; Feces ; Gastroenteritis ; epidemiology ; Genotype ; Humans ; Molecular Epidemiology ; Norovirus ; Phylogeny ; RNA, Viral ; Sequence Analysis, DNA