Investigation of underreporting of death monitoring in Xi’an city, 2018-2020 / 西安交通大学学报(医学版)

【Objective】 To evaluate the underreporting of death cases and related factors in disease surveillance system of Xi’an. 【Methods】 Multi-stage cluster random sampling was used, and 733 villages (neighborhood committees) were selected from 44 townships or streets in Xi’an. All the death cases of the residents in the sampled areas from 2018 to 2020 were collected through a variety of channels and checked with those registered in the Disease Surveillance System. Then the missing cases were investigated and verified. The underreporting rate was calculated, the Excel software was applied for statistical processing and Chi square analysis, and the related factors of underreporting were analyzed by using Logistic regression method. 【Results】 A total of 37650 death cases were investigated from 2018 to 2020; the average mortality was 650.59/105, and 2 901 cases were underreported. From 2018 to 2020, the underreporting rate was 9.89%, 6.95%, and 6.24%, respectively (χ2=133.525, P<0.001), with the average underreporting rate of 7.71%. The underreporting rate in the 44 sampled areas ranged from 0.90% to 42.07%. Multivariate Logistic regression analysis showed that compared with that in rural areas (9.87%), the underreporting rate was lower in urban areas (5.91%, OR=0.567, 95% CI:0.525-0.613), and higher in children under 5 years old (31.20%, OR=5.494, 95% CI:3.732-8.090) and people aged 15-44 years old (11.85%, OR=1.541, 95% CI:1.284-1.846) compared to people over 65 years old (7.44%), and higher in 2018 (9.89%, OR=1.702, 95% CI:1.551-1.869) and 2019 (6.95%, OR=1.148, 95% CI:1.038-1.271) compared to the year 2020 (6.24%). There were 2901 cases reported missing, and the proportion of those who died at home was the highest (81.39%). Underreporting of death more easily occurred in heart diseases (36.80%) and cerebrovascular diseases (27.08%) than others. 【Conclusion】 The reporting completeness of disease surveillance system increased in Xi’an. The overall underreporting rate of death causes decreased with year from 2018 to 2020. The underreporting rates in age group under 5 years old and in rural population were still high. Therefore, the reporting and management of death information should still be strengthened to minimize the underreporting rate.

Evaluation of a Rapid and Accessible RT-qPCR Approach for SARS-CoV-2 Variant of Concern Identification

The ability to distinguish between SARS-CoV-2 variants of concern (VOCs) is of ongoing interest due to differences in transmissibility, response to vaccination, clinical prognosis, and therapy. Although detailed genetic characterization requires whole-genome sequencing (WGS), targeted nucleic acid amplification tests can serve a complementary role in clinical settings, as they are more rapid and accessible than sequencing in most laboratories. We designed and analytically validated a two-reaction multiplex reverse transcription quantitative PCR (RT-qPCR) assay targeting spike protein mutations L452R, E484K, and N501Y in Reaction 1, and del69-70, K417N, and T478K in Reaction 2. This assay had 95-100% agreement with WGS in 502 upper respiratory swabs collected between April 26 and August 1, 2021, consisting of 43 Alpha, 2 Beta, 20 Gamma, 378 Delta, and 59 non-VOC infections. Validation in a separate group of 230 WGS-confirmed Omicron variant samples collected in December 2021 and January 2022 demonstrated 100% agreement. This RT-qPCR-based approach can be implemented in clinical laboratories already performing SARS-CoV-2 nucleic acid amplification tests to assist in local epidemiological surveillance and clinical decision-making.

Concurrent Hearing and Genetic Screening among Newborns in Ningbo, China.

OBJECTIVE: To detect the carrier rates of deafness gene variants in populations in Ningbo and analyze the risk of hereditary hearing loss through concurrent hearing and genetic screening tests. METHODS: Two thousand one hundred and seventy-four newborns were enrolled from November 2018 to August 2019. All subjects underwent hearing screening and newborn deafness genetic screening with 15 variants in 4 genes, and the positive sites were simultaneously verified by sequencing. RESULTS: The total carrier rate of genetic variants in Ningbo reached 4.32%, when GJB2 c.235delC was the variant with the highest prevalence (2.12%), approximately accounting for 48.9% of the total carrier frequency. The carrier frequency of SLC26A4 c.919-2A>G was 0.87%, while the most common variant in mitochondrial DNA (mtDNA) MT-RNR1 gene was m.1555A>G, and its carrier frequency was 0.184%. In the OAE testing, 92 newborns passing hearing screening were tested positively for variants in 4 genes, and 2 of 42 newborns who failed in the first hearing test were found to mutate in 4 genes. CONCLUSION: Herein, the results concerning the carrier rates for deafness gene mutations of Ningbo population are reported. Our study is beneficial to the insight into the deafness genomic epidemiology for deafness genes in Ningbo population and provides the reference for healthcare in Ningbo.

Effect of ORF7 of SARS-CoV-2 on the chemotaxis of monocytes and neutrophils in vitro

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most significant public health threats in worldwide. Patients with severe COVID-19 usually have pneumonia concomitant with local inflammation and sometimes a cytokine storm. Specific components of the SARS-CoV-2 virus trigger lung inflammation, and recruitment of immune cells to the lungs exacerbates this process, although much remains unknown about the pathogenesis of COVID-19. Our study of lung type II pneumocyte cells (A549) demonstrated that ORF7, an open reading frame (ORF) in the genome of SARS-CoV-2, induced the production of CCL2, a chemokine that promotes the chemotaxis of monocytes, and decreased the expression of IL-8, a chemokine that recruits neutrophils. A549 cells also had an increased level of IL-6. The results of our chemotaxis transwell assay suggested that ORF7 augmented monocyte infiltration and reduced the number of neutrophils. We conclude that the ORF7 of SARS-CoV-2 may have specific effects on the immunological changes in tissues after infection. These results suggest that the functions of other ORFs of SARS-CoV-2 should also be comprehensively examined.

Multiplex SARS-CoV-2 Genotyping PCR for Population-Level Variant Screening and Epidemiologic Surveillance

BackgroundEmergence of SARS-CoV-2 variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for pandemic response, but many laboratories do not have the resources to support population-level sequencing. We hypothesized that a spike genotyping nucleic acid amplification test (NAAT) could facilitate high-throughput variant surveillance. MethodsWe designed and analytically validated a one-step multiplex allele-specific reverse transcriptase polymerase chain reaction (RT-qPCR) to detect three non-synonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing. We then screened a large cohort of SARS-CoV-2 positive specimens from our San Francisco Bay Area population. ResultsBetween December 1, 2020 and March 1, 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8%. We detected 1,567 L452R mutations (38.7%), 34 N501Y mutations (0.84%), 22 E484K mutations (0.54%), and 3 (0.07%) E484K+N501Y mutations. The assay had near-perfect (98-100%) concordance with whole-genome sequencing in a validation subset of 229 specimens, and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others. The assay revealed rapid emergence of L452R in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021. ConclusionsWe developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening. This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid. Summary / Key PointsEmergence of SARS-CoV-2 variants with concerning phenotypes is of public health interest. We developed a multiplex genotyping RT-qPCR to rapidly detect L452R, E484K, and N501Y with high sequencing concordance. This high-throughput alternative to resource-intensive sequencing enabled surveillance of L452R emergence.

Persistent SARS-CoV-2 infection and increasing viral variants in children and young adults with impaired humoral immunity

BackgroundThere is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses. MethodsWe describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape. FindingsWe found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape. InterpretationOur results highlight the need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered. FundingThe work was partially funded by The Saban Research Institute at Childrens Hospital Los Angeles intramural support for COVID-19 Directed Research (X.G. and J.D.B.), the Johns Hopkins Center of Excellence in Influenza Research and Surveillance HHSN272201400007C (A.P.), NIH/NIAID R01AI127877 (S.D.B.), NIH/NIAID R01AI130398 (S.D.B.), NIH 1U54CA260517 (S.D.B.), an endowment to S.D.B. from the Crown Family Foundation, an Early Postdoc.Mobility Fellowship Stipend to O.F.W. from the Swiss National Science Foundation (SNSF), and a Coulter COVID-19 Rapid Response Award to S.D.B. L.G. is a SHARE Research Fellow in Pediatric Hematology-Oncology.

Profiling SARS-CoV-2 mutation fingerprints that range from the viral pangenome to individual infection quasispecies

BackgroundThe genome of SARS-CoV-2 is susceptible to mutations during viral replication due to the errors generated by RNA-dependent RNA polymerases. These mutations enable the SARS-CoV-2 to evolve into new strains. Viral quasispecies emerge from de novo mutations that occur in individual patients. In combination, these sets of viral mutations provide distinct genetic fingerprints that reveal the patterns of transmission and have utility in contract tracing. MethodsLeveraging thousands of sequenced SARS-CoV-2 genomes, we performed a viral pangenome analysis to identify conserved genomic sequences. We used a rapid and highly efficient computational approach that relies on k-mers, short tracts of sequence, instead of conventional sequence alignment. Using this method, we annotated viral mutation signatures that were associated with specific strains. Based on these highly conserved viral sequences, we developed a rapid and highly scalable targeted sequencing assay to identify mutations, detect quasispecies and identify mutation signatures from patients. These results were compared to the pangenome genetic fingerprints. ResultsWe built a k-mer index for thousands of SARS-CoV-2 genomes and identified conserved genomics regions and landscape of mutations across thousands of virus genomes. We delineated mutation profiles spanning common genetic fingerprints (the combination of mutations in a viral assembly) and rare ones that occur in only small fraction of patients. We developed a targeted sequencing assay by selecting primers from the conserved viral genome regions to flank frequent mutations. Using a cohort of SARS-CoV-2 clinical samples, we identified genetic fingerprints consisting of strain-specific mutations seen across populations and de novo quasispecies mutations localized to individual infections. We compared the mutation profiles of viral samples undergoing analysis with the features of the pangenome. ConclusionsWe conducted an analysis for viral mutation profiles that provide the basis of genetic fingerprints. Our study linked pangenome analysis with targeted deep sequenced SARS-CoV-2 clinical samples. We identified quasispecies mutations occurring within individual patients, mutations demarcating dominant species and the prevalence of mutation signatures, of which a significant number were relatively unique. Analysis of these genetic fingerprints may provide a way of conducting molecular contact tracing.

SARS-CoV-2 Antibody Responses Correlate with Resolution of RNAemia But Are Short-Lived in Patients with Mild Illness

SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, could offer protective immunity, and may affect clinical outcomes of COVID-19 patients. We analyzed 625 serial plasma samples from 40 hospitalized COVID-19 patients and 170 SARS-CoV-2-infected outpatients and asymptomatic individuals. Severely ill patients developed significantly higher SARS-CoV-2-specific antibody responses than outpatients and asymptomatic individuals. The development of plasma antibodies was correlated with decreases in viral RNAemia, consistent with potential humoral immune clearance of virus. Using a novel competition ELISA, we detected antibodies blocking RBD-ACE2 interactions in 68% of inpatients and 40% of outpatients tested. Cross-reactive antibodies recognizing SARS-CoV RBD were found almost exclusively in hospitalized patients. Outpatient and asymptomatic individuals serological responses to SARS-CoV-2 decreased within 2 months, suggesting that humoral protection may be short-lived.

High-throughput SARS-CoV-2 and host genome sequencing from single nasopharyngeal swabs

During COVID19 and other viral pandemics, rapid generation of host and pathogen genomic data is critical to tracking infection and informing therapies. There is an urgent need for efficient approaches to this data generation at scale. We have developed a scalable, high throughput approach to generate high fidelity low pass whole genome and HLA sequencing, viral genomes, and representation of human transcriptome from single nasopharyngeal swabs of COVID19 patients.

Targeted identification of C-type lectins in snake venom by 2DE and Western blot.

C-type lectins (CTL) and CTL-like proteins (snaclecs) are important toxins found in snake venom which can disrupt hemostasis by binding platelet membrane glycoproteins. Traditional identification of these toxins usually relies on an "activity-directed fractionation" approach which is very arduous. Here, we report a new method for rapid screening of these proteins in snake venom. METHODS: A conserved and immunogenic peptide found in svCTLs (CTL and snaclecs) was identified by sequence alignment using DNAStar software. The peptide was de novo synthesized and conjugated to keyhole limpet hemocyanin (KLH). Rabbit antibodies were generated against the peptide by classical immunization. Deinagkistrodon acutus venom was separated by two-dimensional electrophoresis (2DE) followed by Western blot and CTLs immunodetected using the isolated polyclonal antibody. The same svCTL spots on a parallel 2DE gel were isolated and analyzed by MALDI-TOF-MS. RESULTS: A highly conserved peptide with the sequence "KTWDDAEKFCTEQ" was identified as a common epitope in svCTLs. The polyclonal antibody against the 13aa-peptide was successfully prepared and purified. Its usefulness to detect svCTLs in D. acutus venom was tested by 2DE-WB and we determined that it positively identified all known D. acutus venom CTLs. CONCLUSIONS: Immunodetection with antibodies against KTWDDAEKFCTEQ is an efficient strategy to identify novel svCTLs in the context of a complex proteome.

Retrospective Pooled Screening for SARS-CoV-2 RNA in late 2019

Reports have emerged documenting earlier SARS-CoV-2 cases than previously recognized. To investigate this possibility in the Bay Area, we retrospectively tested 1,700 samples from symptomatic individuals for the last 2 months of 2019. No SARS-CoV-2 positive pools were identified, consistent with limited transmission in this population at this time.

High frequency of SARS-CoV-2 RNAemia and association with severe disease

BackgroundDetection of SARS-CoV-2 RNA in the blood, also known as RNAemia, has been reported, but its prognostic implications are not well understood. This study aimed to determine the frequency of SARS-CoV-2 RNA in plasma and its association with the clinical severity of COVID-19. MethodsAn analytical cross-sectional study was performed in a single-center tertiary care institution in northern California and included consecutive inpatients and outpatients with COVID-19 confirmed by detection of SARS-CoV-2 RNA in nasopharyngeal swab specimens. The prevalence of SARS CoV-2 RNAemia and the strength of its association with clinical severity variables were examined and included the need for transfer to an intensive care unit (ICU), mechanical ventilation and 30-day all-cause mortality. ResultsPaired nasopharyngeal and plasma samples were included from 85 patients. The overall median age was 55 years, and individuals with RNAemia were older than those with undetectable SARS-CoV-2 RNA in plasma (63 vs 50 years; p=0.001). Comorbidities were frequent including obesity (37.7%), hypertension (30.6%) and diabetes mellitus (22.4%). RNAemia was detected in a total of 28/85 (32.9%) individual patients, including 22/28 (78.6%) who required hospital admission. RNAemia was detected more frequently in individuals who developed severe disease including the need for ICU transfer (32.1% vs 14.0%; p=0.05), mechanical ventilation (21.4% vs 3.5%; p=0.01) and 30-day all-cause mortality (14.3% vs 0%; p=0.01). No association was detected between RNAemia and estimated levels of viral RNA in the nasopharynx. An additional 121 plasma samples from 28 individuals with RNAemia were assessed longitudinally, and RNA was detected for a maximum duration of 10 days. ConclusionThis study demonstrated a high proportion of SARS-CoV-2 RNAemia, and an association between RNAemia and clinical severity suggesting the potential utility of plasma viral testing as a prognostic indicator for COVID-19.

Comparison of the Accula SARS-CoV-2 Test with a Laboratory-Developed Assay for Detection of SARS-CoV-2 RNA in Clinical Nasopharyngeal Specimens

BackgroundSeveral point-of-care (POC) molecular tests have received emergency use authorization (EUA) from the Food and Drug Administration (FDA) for diagnosis of SARS-CoV-2. The test performance characteristics of the Accula (Mesa Biotech) SARS-CoV-2 POC test need to be evaluated to inform its optimal use. ObjectivesThe aim of this study was to assess test performance of the Accula SARS-CoV-2 test. Study designThe performance of the Accula test was assessed by comparing results of 100 nasopharyngeal swab samples previously characterized by the Stanford Health Care EUA laboratory-developed test (SHC-LDT) targeting the envelope (E) gene. Assay concordance was assessed by overall percent agreement, positive percent agreement (PPA), negative percent agreement (NPA), and Cohens kappa coefficient. ResultsOverall percent agreement between the assays was 84.0% (95% confidence interval [CI] 75.3 to 90.6%), PPA was 68.0% (95% CI 53.3 to 80.5%) and the kappa coefficient was 0.68 (95% CI 0.54 to 0.82). Sixteen specimens detected by the SHC-LDT were not detected by the Accula test, and showed low viral load burden with a median cycle threshold value of 37.7. NPA was 100% (95% CI 94.2 to 100%). ConclusionCompared to the SHC-LDT, the Accula SARS-CoV-2 test showed excellent negative agreement. However, positive agreement was low for samples with low viral load. The false negative rate of the Accula POC test calls for a more thorough evaluation of POC test performance characteristics in clinical settings, and for confirmatory testing in individuals with moderate to high pre-test probability of SARS-CoV-2 who test negative on Accula.

The relationship between PCT,CRP and BNP levels in peripheral blood of patients with sepsis and cTn I and its clinical significance / 国际检验医学杂志

Objective To investigate the relationship between serum PCT,CRP,BNP and cardiac troponin I (cTnI)and its clinical significance in patients with sepsis.Methods 112 patients with sepsis treated in our hospital were collected.Patients were divided into sepsis,severe sepsis and septic shock groups according to their degree of illness.The serum levels of cTnI and cTnI were measured in peripheral blood samples of pa-tients with sepsis,severe sepsis and sepsis shock.To analyze the correlation between serum cTnI and cTn I in patients with sepsis.The levels of BNP and cTnI were significantly higher in septic shock group than in severe sepsis group.The difference was statistically significant(P<0.01),and that in severe sepsis group was signif-icantly higher than that in sepsis group(P<0.01).The results of correlation analysis showed that there was a positive correlation between serum PCT,BNP,CRP and cTnI levels,but the correlation was PCT(r =0.831)>BNP(r=0.456)>CRP(r=0.287).Results Conclusion cTnI and serum PCT,CRP,BNP are in the serum of the patients.With the exacerbation of sepsis patients,there will be more significant myocardial injury symptoms.

Correlation between chronic hepatitis B and serum triglyceride and cholesterol levels / 中华传染病杂志

Objective To investigate the relationship between various stages of chronic hepatitis B virus (HBV) infection and lipid metabolism and its influencing factors.Methods Seventy-two cases of chronic hepatitis B (CHB),40 cases of liver cirrhosis and 17 cases of hepatocellular carcinoma (HCC) were enrolled.One-way ANOVA analyses were used to compare age,gender,liver function,lipid metabolism,and HBV DNA levels of each group.Pearson correlation analysis was used to study the correlation between HBV DNA and lipid metabolism.Binary Logistic regression analyses were performed to explore the risk factors of cirrhosis and HCC in patients with CHB.Results Differences of age,alanine aminotransferase (ALT),albumin (Alb),triglyceride (TG),and cholesterol(CHO) among the three groups (CHB group,cirrhosis group and HCC group) were statistically significant (all P＜0.05).TG levels in cirrhosis and HCC groups were (-0.061± 0.234)lg mmol/L and (-0.061±0.253) lg mmol/L,respectively,which were both significantly lower than that of the CHB group (0.116±0.182) lg mmol/L (F=11.466,P=0.000).CHO level in cirrhosis group was (0.460±0.333) lg mmol/L,which was lower than that in CHB group (0.586±0.101) lg mmol/L (F=4.892,P=0.009).The HBV DNA levels inversely correlated with TG and CHO levels in CHB group (r=-0.266,P=0.024; r=-0.309,P=0.008,respectively).The HBV DNA levels of cirrhosis and HCC patients positively correlated with ALT levels (r=0.355,P =0.007).Old age (OR=1.096,95％CI:1.025-1.172),low Alb (OR=0.000,95％CI:0.000-0.000),and low levels of ALT (OR=0.128,95％CI:0.026-0.641) were risk factors for development of cirrhosis and HCC in CHB patients (all P＜0.05).Conclusions With the progression of liver injuries,TG and CHO levels are reduced.Further studies of correlation between risk factors for the development of cirrhosis and HCC and lipid metabolism in CHB patients are needed.

Analysis of Death Cause of Children between New Born and Fourteen Years of Age from 2005 to 2007 in Xi'an City / 中国慢性病预防与控制

Objective To investigate the characteristic of death in children between the new born and fourteen years of age in Xi'an City. Methods Surveillance data on resident death from 2005 to 2007 in Xi'an City was analyzed, classified the death cause with ICD-10. Results The average annual mortality rate in children between new born and fourteen years of age in our city was 97.91/100 000 (accounted for 3.1% of the total death), mortality rate in boys (114.39/100 000) was higher than that in girls (79.33/100 000), mortality rate in infants within one year of age was the highest (818.38 / 100 000), neonatal mortality rate counted for 83.65% of infant death; Injury, some diseases originated from perinatal period and congenital malformation were three main causes of children death. Conclusion Injury, some diseases originated from perinatal period and congenital malformation are three main causes of children death; Invention to injury and premarital, maternal health care are needed.