Proteomic and Cellular Characterization of Omicron Breakthrough Infections and a Third Homologous or Heterologous Boosting Vaccination in a Longitudinal Cohort.

The understanding of dynamic plasma proteome features in hybrid immunity and breakthrough infection is limited. A deeper understanding of the immune differences between heterologous and homologous immunization could assist in the future establishment of vaccination strategies. In this study, 40 participants who received a third dose of either a homologous BBIBP-CorV or a heterologous ZF2001 protein subunit vaccine following two doses of inactivated coronavirus disease 2019 vaccines and 12 patients with BA2.2 breakthrough infections were enrolled. Serum samples were collected at days 0, 28, and 180 following the boosting vaccination and breakthrough and then analyzed using neutralizing antibody tests and mass spectrometer-based proteomics. Mass cytometry of peripheral blood mononuclear cell samples was also performed in this cohort. The chemokine signaling pathway and humoral response markers (IgG2 and IgG3) associated with infection were found to be upregulated in breakthrough infections compared to vaccination-induced immunity. Elevated expression of IGKV, IGHV, IL-17 signaling, and the phagocytosis pathway, along with lower expression of FGL2, were correlated with higher antibody levels in the boosting vaccination groups. The MAPK signaling pathway and Fc gamma R-mediated phagocytosis were more enriched in the heterologous immunization groups than in the homologous immunization groups. Breakthrough infections can trigger more intensive inflammatory chemokine responses than vaccination. T-cell and innate immune activation have been shown to be closely related to enhanced antibody levels after vaccination and therefore might be potential targets for vaccine adjuvant design.

Clinical Characteristics, Treatment, and Prognosis of Osteoarticular Brucellosis: A Retrospective Real-World Study in Shenyang, China, 2014-2019.

This study aimed to assess the clinical characteristics, treatment, and prognosis of osteoarticular brucellosis. We conducted a retrospective study enrolling brucellosis patients from the Sixth People's Hospital of Shenyang between September 2014 and June 2019. A total of 1917 participants were admitted during this period. After applying propensity score matching, we retrospectively analyzed 429 patients with osteoarthritis and 429 patients without osteoarthritis. The primary outcome was treatment completion. The secondary outcome was symptom disappearance and seroconversion. Brucellosis patients with osteoarthritis had longer treatment course (160 [134.3-185.7] vs. 120 [102.3-137.7] d, p = 0.008) than those without osteoarthritis. The most common involved site was lumbar vertebrae (290 [67.6%]) in brucellosis patients with osteoarthritis. Longer symptom duration (90 [83.0-97.0] vs. 42 [40.2-43.8], p < 0.001) along with no significant difference in seroconversion (180 [178.8-181.2] vs. 180 [135.1-224.9], p = 0.212) was observed in osteoarthritis patients with treatment course >90 d. Peripheral joint involvement (adjusted hazard ratio [95% confidence interval] 1.485 [1.103-1.999]; p = 0.009) had a shorter symptom duration compared with shaft joint involvement. No significant differences were observed in treatment therapy between doxycycline plus rifampin (DR) or plus cephalosporins (DRC) in treatment course (p = 0.190), symptom persistence (p = 0.294), and seroconversion (p = 0.086). Lumbar vertebra was the most commonly involved site. Even if all symptoms disappeared, Serum agglutination test potentially remained positive in some patients. Compared with peripheral arthritis, shaft arthritis was the high-risk factor for longer symptom duration. The therapeutic effects were similar between DR and DRC. In summary, our study provided important insights into the clinical characteristics, treatment, and outcomes of osteoarticular brucellosis. Clinical Trial Registration number: NCT04020536.

Natural History of and Dynamic Changes in Clinical Manifestation, Serology, and Treatment of Brucellosis, China.

Serum agglutination test plus exposure history were used to diagnose most cases of human brucellosis in 2 China provinces. After appropriate treatment, 13.3% of acute brucellosis cases progressed to chronic disease; arthritis was an early predictor. Seropositivity can persist after symptoms disappear, which might cause physicians to subjectively extend therapeutic regimens.

Risk of reinfection and severity with the predominant BA.5 Omicron subvariant China, from December 2022 to January 2023.

Data on reinfection in large Asian populations are limited. In this study, we aimed to evaluate the reinfection rate, disease severity, and time interval between the infections in the symptomatic and asymptomatic populations which are firstl infected with BA.2 Omicron Variant. We retrospectively included adult patients with COVID-19 discharged from four designated hospitals between 27 April 2021 and 30 November 2022, who were interviewed via telephone from 29 January to 1 March 2023. Univariable and multivariable analyses were used to explore risk factors associated with reinfection. A total of 16,558 patients were followed up, during the telephone survey of an average of 310.0 days, 1610 (9.72%) participants self-reported reinfection. The mean time range of reinfection was 257.9 days. The risks for reinfection were analysed using multivariable logistic regression. Patients with severe first infection were at higher risk for reinfection (aORs, 2.50; P < 0.001). The male (aORs,0.82; P < 0.001), the elderly (aORs, 0.44; P < 0.001), and patients with full vaccination (aORs, 0.67; P < 0.001) or booster (aORs, 0.63; P < 0.001) had the lower risk of reinfection. Patients over 60 years of age (aORs,9.02; P = 0.006) and those with ≥2 comorbidities (aORs,11.51; P = 0.016). were at higher risk for severe reinfection. The number of clinical manifestations of reinfection increases in people with severe first infection (aORs, 2.82; P = 0.023). The overall reinfection rate was 9.72%, and the reinfection rate of Omicron-to-Omicron subvariants was 9.50% at one year. The severity of Omicron-Omicron reinfection decreased. Data from our clinical study may provide clinical evidence and bolster response preparedness for future COVID-19 reinfection waves.

Utility of clinical metagenomics in diagnosing malignancies in a cohort of patients with Epstein-Barr virus positivity.

Backgrounds: Differentiation between benign and malignant diseases in EBV-positive patients poses a significant challenge due to the lack of efficient diagnostic tools. Metagenomic Next-Generation Sequencing (mNGS) is commonly used to identify pathogens of patients with fevers of unknown-origin (FUO). Recent studies have extended the application of Next-Generation Sequencing (NGS) in identifying tumors in body fluids and cerebrospinal fluids. In light of these, we conducted this study to develop and apply metagenomic methods to validate their role in identifying EBV-associated malignant disease. Methods: We enrolled 29 patients with positive EBV results in the cohort of FUO in the Department of Infectious Diseases of Huashan Hospital affiliated with Fudan University from 2018 to 2019. Upon enrollment, these patients were grouped for benign diseases, CAEBV, and malignant diseases according to their final diagnosis, and CNV analysis was retrospectively performed in 2022 using samples from 2018 to 2019. Results: Among the 29 patients. 16 of them were diagnosed with benign diseases, 3 patients were diagnosed with CAEBV and 10 patients were with malignant diseases. 29 blood samples from 29 patients were tested for mNGS. Among all 10 patients with malignant diagnosis, CNV analysis suggested neoplasms in 9 patients. Of all 19 patients with benign or CAEBV diagnosis, 2 patients showed abnormal CNV results. The sensitivity and specificity of CNV analysis for the identification for tumors were 90% and 89.5%, separately. Conclusions: The application of mNGS could assist in the identification of microbial infection and malignancies in EBV-related diseases. Our results demonstrate that CNV detection through mNGS is faster compared to conventional oncology tests. Moreover, the convenient collection of peripheral blood samples adds to the advantages of this approach.

Pathogen quantitative efficacy of different spike-in internal controls and clinical application in central nervous system infection with metagenomic sequencing.

IMPORTANCE: Metagenomic next-generation sequencing (mNGS) has been used broadly for pathogens detection of infectious diseases. However, there is a lack of method for the absolute quantitation of pathogens by mNGS. We compared the quantitative efficiency of three mNGS internal controls (ICs) Thermus thermophilus, T1 phages, and artificial DNA sequence and developed the most applicable strategies for pathogen quantitation via mNGS in central nervous system infection. The IC application strategy we developed will enable mNGS analysis to assess the pathogen load simultaneously with the detection of pathogens, which should provide critical information for quick decision-making of treatment as well as clinical prognosis.

Clinical Diagnostic Performance of Droplet Digital PCR for Suspected Bloodstream Infections.

Accurate and timely etiological diagnosis is crucial for bloodstream infections (BSIs) due to their high disability and mortality. We conducted a single-center prospective cohort study to compare the digital droplet PCR (ddPCR) assay with traditional blood culture. A total of 169 blood samples from 122 patients with suspected BSIs were collected, mostly from the department of infectious diseases, the emergency department, and the intensive care units, and the clinical data were also recorded. Nucleic acid was extracted from the blood samples, and a 5-fluorescent-channel droplet digital PCR assay was performed and then fed back with the pathogen and its copies. In BSI patients, ddPCR reported an overall 85.71% (12/14) (95% confidence interval [CI], 56.15 to 97.48%) sensitivity, 100% (7/7) (95% CI, 56.09 to 100.00%) and 71.43% (5/7) (95% CI, 30.26 to 94.89%) sensitivity in patients without empirical treatment and in empirically treated patients, respectively. Compared to traditional blood culture, the overall detection rate of ddPCR was significantly higher, 11.27% (16/142) (95% CI, 6.78 to 17.93%) versus 30.28% (43/142) (95% CI, 23.01 to 38.64%), and the extra detection rate of ddPCR was 19.01% (27/142) (95% CI, 13.11 to 26.63%). Of the ddPCR-positive culture-negative cases, 74.19% (23/31) (95% CI, 55.07 to 87.46%) were consistent with the final clinical diagnosis, including 10 bacteria and fungi. The detection rate of ddPCR was significantly higher in patients with white blood cell (WBC) counts of >10 · 109/L, C-reactive protein (CRP) of >70 mg/L, or procalcitonin (PCT) of >0.9 ng/L. Pathogen loads detected by ddPCR are correlated with WBC, CRP, and especially, PCT levels, precisely and rapidly reflecting clinical disease progression. ddPCR has an important guiding value for the clinical use of antibiotics to achieve the best pathogen coverage and the antibacterial effect. Collectively, ddPCR showed a great diagnostic performance in BSIs and had an overall higher detection rate than blood culture. In addition, ddPCR could be used to dynamically monitor the disease progression and provide medication guidance on antibiotic use. IMPORTANCE ddPCR is a promising method to address the current challenges of BSI diagnosis and precise treatment, as it is highly efficient in DNA detection. It shortens the identification of BSI-related pathogens from several days of traditional bacterial culture to 4 to 5 h. It is extremely sensitive and more tolerant to PCR inhibitors, which may facilitate the amplification and enable the detection of a meager amount of DNA fragments in detecting BSI-related pathogens and drug-resistant genes. It can identify almost 20 pathogens in one reaction, which reduces the usage of clinical blood samples to no more than 2 mL. Additionally, dynamic monitoring, assessment of pathogens, and antibiotic resistance genes in patients could be used to guide timely and precise adjustment of antimicrobial prescription. The short turnaround time of ddPCR may have the potential to guide antimicrobial treatment in the very early stage of sepsis and reduce the mortality and disability rate of sepsis.

Clinical application and drug-use-guidance value of metagenomic next-generation sequencing in central nervous system infection.

OBJECTIVE: Timely and precise etiology diagnosis is crucial for optimized medication regimens and better prognosis in central nervous system infections (CNS infections). We aimed to analyze the impact of mNGS tests on the management of patients with CNS infections. METHODS: We conducted a single-center retrospective cohort study to analyze the value of mNGS in clinical applications. Three hundred sixty-nine patients with a CNS infection diagnosis were enrolled, and their clinical data were collected. CDI and DDI were defined in our study to describe the intensity of drug use in different groups. We used LOH and mRS to evaluate if the application of mNGS can benefit CNS infected patients. RESULTS: mNGS reported a 91.67% sensitivity in culture-positive patients and an 88.24% specificity compared with the final diagnoses. Patients who participated with the mNGS test had less drug use, both total (58.77 vs. 81.18) and daily (22.6 vs. 28.12, P < 0.1, McNemar) intensity of drug use, and length of hospitalization (23.14 vs. 24.29). Patients with a consciousness grading 1 and 3 had a decrease in CDI (Grade 1, 86.49 vs. 173.37; Grade 3, 48.18 vs. 68.21), DDI (Grade 1, 1.52 vs. 2.72; Grade 3, 2.3 vs. 2.45), and LOH (Grade 1, 32 vs. 40; Grade 3, 21 vs. 23) with the application of mNGS. Patients infected with bacteria in the CNS had a reduced CDI, DDI, and LOH in the mNGS group. This was compared with the TraE group that had 49% of patients altered medication plans, and 24.7% of patients reduced drug intensity four days after mNGS reports. This was because of the reduction of drug types. CONCLUSION: mNGS showed its high sensitivity and specificity characteristics. mNGS may assist clinicians with more rational medication regimens and reduce the drug intensity for patients. The primary way of achieving this is to reduce the variety of drugs, especially for severe patients and bacterial infections. mNGS has the ability of improving the prognosis of CNS infected patients.

Safety and immunogenicity of a third-dose homologous BBIBP-CorV boosting vaccination: interim results from a prospective open-label study.

A COVID-19 booster vaccination is being comprehensively evaluated globally due to the emerging concern of reduced protection rate of previous vaccination and circulating Variants of Concern (VOC). But the safety and immunogenicity of homologous BBIBP-CorV boosting vaccination are yet to be thoroughly evaluated. We conducted this prospective, open-label study in Huashan Hospital using a third 6.5U BBIBP-CorV administered at an interval of 4-8 months following the previous two doses in healthy adults. Safety, anti-RBD response and neutralizing titers against SARS-CoV-2 and VOCs were examined. Sixty-three and forty participants entered the booster and the control group, respectively. A significant increase in IFN-γ SFU per million PBMCs was observed on day 14 against N peptide (20 vs. 5, P < 0.001). On day 14, pVNT GMTs increased over 15 folds of the baseline levels against prototype to reach 404.54 titers and over 9-13 folds against 4 VOCs and continuously increased by day 28. sVNT GMTs increased 112.51 and 127.45 folds by days 14 and 28 compared to the baseline level. Median anti-RBD antibody and IgG level significantly increased from 11.12 to 2607.50 BAU/ml and 4.07 to 619.20 BAU/ml on day 14. On day 14, females showed a significantly higher cell-mediated immune response against S1 peptide. The 7-8 months interval group had a higher humoral response than the 4-6 months interval group. No severe adverse event was reported. A third homologous BBIBP-CorV boosting vaccination was safe and highly immunogenic for healthy adults and broadened participants' immunity against VOCs.

A diagnostic accuracy study comparing RNA LAMP, direct LAMP, and rapid antigen testing from nasopharyngeal swabs.

Introduction: During the coronavirus disease 2019 (COVID-19) pandemic, the early detection and isolation of individuals infected with severe acute respiratory syndrome coronavirus disease 2 (SARS-CoV-2) through mass testing can effectively prevent disease transmission. SARS-CoV-2 nucleic acid rapid detection based on loop-mediated isothermal amplification (LAMP) may be appropriate to include in testing procedures. Methods: We used 860 nasopharyngeal specimens from healthcare workers of Huashan Hospital and COVID-19 patients collected from April 7th to 21st, 2022, to assess the clinical diagnostic performance of the LAMP assay marketed by Shanghai GeneSc Biotech and compared it to the result of a rapid antigen test (RAT) head-to-head. Results: Overall, the diagnostic performance of LAMP assay and RAT were as follows. The LAMP assay represented higher sensitivity and specificity than RAT, especially in the extracted RNA samples. The sensitivity was 70.92% and 92.91% for direct LAMP and RNA-LAMP assay, respectively, while the specificity was 99.86% and 98.33%. The LAMP assay had overall better diagnostic performance on the specimens with relatively lower C t values or collected in the early phase (≤7 days) of COVID-19. The combination of LAMP assay and RAT improved diagnostic efficiency, providing new strategies for rapidly detecting SARS-CoV-2. Conclusion: The LAMP assay are suitable for mass screenings of SARS-CoV-2 infections in the general population.

SARS-CoV-2 intra-host single-nucleotide variants associated with disease severity.

Variants of severe acute respiratory syndrome coronavirus 2 frequently arise within infected individuals. Here, we explored the level and pattern of intra-host viral diversity in association with disease severity. Then, we analyzed information underlying these nucleotide changes to infer the impetus including mutational signatures and immune selection from neutralizing antibody or T-cell recognition. From 23 January to 31 March 2020, a set of cross-sectional samples were collected from individuals with homogeneous founder virus regardless of disease severity. Intra-host single-nucleotide variants (iSNVs) were enumerated using deep sequencing. Human leukocyte antigen (HLA) alleles were genotyped by Sanger sequencing. Medical records were collected and reviewed by attending physicians. A total of 836 iSNVs (3-106 per sample) were identified and distributed in a highly individualized pattern. The number of iSNVs paced with infection duration peaked within days and declined thereafter. These iSNVs did not stochastically arise due to a strong bias toward C > U/G > A and U > C/A > G substitutions in reciprocal proportion with escalating disease severity. Eight nonsynonymous iSNVs in the receptor-binding domain could escape from neutralization, and eighteen iSNVs were significantly associated with specific HLA alleles. The level and pattern of iSNVs reflect the in vivo viral-host interaction and the disease pathogenesis.

Omicron variant showed lower neutralizing sensitivity than other SARS-CoV-2 variants to immune sera elicited by vaccines after boost.

ABSTRACTThe emerging new VOC B.1.1.529 (Omicron) variant has raised serious concerns due to multiple mutations, reported significant immune escape, and unprecedented rapid spreading speed. Currently, studies describing the neutralization ability of different homologous and heterologous booster vaccination against Omicron are still lacking. In this study, we explored the immunogenicity of COVID-19 breakthrough patients, BBIBP-CorV homologous booster group and BBIBP-CorV/ZF2001 heterologous booster group against SARS-CoV-2 pseudotypes corresponding to the prototype, Beta, Delta, and the emergent Omicron variant.Notably, at 14 days post two-dose inactivated vaccines, pVNT titre increased to 67.4 GMTs against prototype, 8.85 against Beta and 35.07 against Delta, while neutralization activity against Omicron was below the lower limit of quantitation in 80% of the samples. At day 14 post BBIBP-CorV homologous booster vaccination, GMTs of pVNT significantly increased to 285.6, 215.7, 250.8, 48.73 against prototype, Beta, Delta, and Omicron, while at day 14 post ZF2001 heterologous booster vaccination, GMTs of pVNT significantly increased to 1436.00, 789.6, 1501.00, 95.86, respectively. Post booster vaccination, 100% samples showed positive neutralization activity against Omicron, albeit illustrated a significant reduction (5.86- to 14.98-fold) of pVNT against Omicron compared to prototype at 14 days after the homologous or heterologous vaccine boosters.Overall, our study demonstrates that vaccine-induced immune protection might more likely be escaped by Omicron compared to prototypes and other VOCs. After two doses of inactivated whole-virion vaccines as the "priming" shot, a third heterologous protein subunit vaccine and a homologous inactivated vaccine booster could improve neutralization against Omicron.

B cell receptor signatures associated with strong and poor SARS-CoV-2 vaccine responses.

Breakthrough infection of SARS-CoV-2 is a serious challenge, as increased infections were documented in fully-vaccinated individuals. Recipients with poor antibody response are highly vulnerable to reinfection, whereas those with strong antibody responses achieve sterilizing immunity. Thus far, biomarkers associated with levels of vaccine-elicited antibody response are still lacking. Here, we studied the antibody response of age- and gender-controlled healthy cohort, who received inactivated SARS-CoV-2 vaccines and profiled the B cell receptor repertoires in longitudinally consecutive samples. Upon vaccination, all vaccinated individuals displayed a convergent antibody response with shared common antibody clones and public neutralizing antibodies. Strikingly, poor vaccine-responders are distinguishable from strong vaccine-responders by a biased V-usage before vaccination and IgG to IgM mRNA ratio. These findings reveal molecular signatures associated with the different levels of vaccine-induced antibody response, which could be further developed into biomarkers for the design of vaccination strategies.

Cellular basis of enhanced humoral immunity to SARS-CoV-2 upon homologous or heterologous booster vaccination analyzed by single-cell immune profiling.

SARS-CoV-2 vaccine booster dose can induce a robust humoral immune response, however, its cellular mechanisms remain elusive. Here, we investigated the durability of antibody responses and single-cell immune profiles following booster dose immunization, longitudinally over 6 months, in recipients of a homologous BBIBP-CorV/BBIBP-CorV or a heterologous BBIBP-CorV/ZF2001 regimen. The production of neutralizing antibodies was dramatically enhanced by both booster regimens, and the antibodies could last at least six months. The heterologous booster induced a faster and more robust plasmablast response, characterized by activation of plasma cells than the homologous booster. The response was attributed to recall of memory B cells and the de novo activation of B cells. Expanded B cell clones upon booster dose vaccination could persist for months, and their B cell receptors displayed accumulated mutations. The production of antibody was positively correlated with antigen presentation by conventional dendritic cells (cDCs), which provides support for B cell maturation through activation and development of follicular helper T (Tfh) cells. The proper activation of cDC/Tfh/B cells was likely fueled by active energy metabolism, and glutaminolysis might also play a general role in promoting humoral immunity. Our study unveils the cellular mechanisms of booster-induced memory/adaptive humoral immunity and suggests potential strategies to optimize vaccine efficacy and durability in future iterations.

Evaluations of Clinical Utilization of Metagenomic Next-Generation Sequencing in Adults With Fever of Unknown Origin.

INTRODUCTION: The diagnosis of infection-caused fever of unknown origin (FUO) is still challenging, making it difficult for physicians to provide an early effective therapy. Therefore, a novel pathogen detection platform is needed. Metagenomic next-generation sequencing (mNGS) provides an unbiased, comprehensive technique for the sequence-based identification of pathogenic microbes, but the study of the diagnostic values of mNGS in FUO is still limited. METHODS: In a single-center retrospective cohort study, 175 FUO patients were enrolled, and clinical data were recorded and analyzed to compare mNGS with culture or traditional methods including as smears, serological tests, and nucleic acid amplification testing (NAAT) (traditional PCR, Xpert MTB/RIF, and Xpert MTB/RIF Ultra). RESULTS: The blood mNGS could increase the overall rate of new organisms detected in infection-caused FUO by roughly 22.9% and 19.79% in comparison to culture (22/96 vs. 0/96; OR, ∞; p = 0.000) and conventional methods (19/96 vs. 3/96; OR, 6.333; p = 0.001), respectively. Bloodstream infection was among the largest group of those identified, and the blood mNGS could have a 38% improvement in the diagnosis rate compared to culture (19/50 vs. 0/50; OR, ∞; p = 0.000) and 32.0% compared to conventional methods (16/50 vs. 3/50; OR, 5.333; p = 0.004). Among the non-blood samples in infection-caused FUO, we observed that the overall diagnostic performance of mNGS in infectious disease was better than that of conventional methods by 20% (9/45 vs. 2/45; OR, 4.5; p = 0.065), and expectedly, the use of non-blood mNGS in non-bloodstream infection increased the diagnostic rate by 26.2% (8/32 vs. 0/32; OR, ∞; p = 0.008). According to 175 patients' clinical decision-making, we found that the use of blood mNGS as the first-line investigation could effectively increase 10.9% of diagnosis rate of FUO compared to culture, and the strategy that the mNGS of suspected parts as the second-line test could further benefit infectious patients, improving the diagnosis rate of concurrent infection by 66.7% and 12.5% in non-bloodstream infection, respectively. CONCLUSION: The application of mNGS in the FUO had significantly higher diagnostic efficacy than culture or other conventional methods. In infection-caused FUO patients, application of blood mNGS as the first-line investigation and identification of samples from suspected infection sites as the second-line test could enhance the overall FUO diagnosis rate and serve as a promising optimized diagnostic protocol in the future.