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.

Early identification and severity prediction of acute respiratory infection (ESAR): a study protocol for a randomized controlled trial.

BACKGROUND: The outbreak of SARS-CoV-2 at the end of 2019 sounded the alarm for early inspection on acute respiratory infection (ARI). However, diagnosis pathway of ARI has still not reached a consensus and its impact on prognosis needs to be further explored. METHODS: ESAR is a multicenter, open-label, randomized controlled, non-inferiority clinical trial on evaluating the diagnosis performance and its impact on prognosis of ARI between mNGS and multiplex PCR. Enrolled patients will be divided into two groups with a ratio of 1:1. Group I will be directly tested by mNGS. Group II will firstly receive multiplex PCR, then mNGS in patients with severe infection if multiplex PCR is negative or inconsistent with clinical manifestations. All patients will be followed up every 7 days for 28 days. The primary endpoint is time to initiate targeted treatment. Secondary endpoints include incidence of significant events (oxygen inhalation, mechanical ventilation, etc.), clinical remission rate, and hospitalization length. A total of 440 participants will be enrolled in both groups. DISCUSSION: ESAR compares the efficacy of different diagnostic strategies and their impact on treatment outcomes in ARI, which is of great significance to make precise diagnosis, balance clinical resources and demands, and ultimately optimize clinical diagnosis pathways and treatment strategies. Trial registration Clinicaltrial.gov, NCT04955756, Registered on July 9th 2021.

Evaluation of droplet digital PCR rapid detection method and precise diagnosis and treatment for suspected sepsis (PROGRESS): a study protocol for a multi-center pragmatic randomized controlled trial.

BACKGROUND: Sepsis is still a major public health concern and a medical emergency due to its high morbidity and mortality. Accurate and timely etiology diagnosis is crucial for sepsis management. As an emerging rapid and sensitive pathogen detection tool, digital droplet PCR (ddPCR) has shown promising potential in rapid identification of pathogens and antimicrobial resistance genes. However, the diagnostic value and clinical impact of ddPCR tests remains to be studied in patients with suspected sepsis. PROGRESS trial is aimed to evaluate the clinical effectiveness of a novel ddPCR assay compared with standard practice. METHODS: PROGRESS is a multicenter, open-label, pragmatic randomized controlled trial (pRCT) set in ten hospitals, including departments of infectious disease and intensive care units. In this study, a total of 2292 patients with suspected sepsis will be randomly assigned to two arms: the ddPCR group and the control group with a ratio of 3:1. The primary outcome is the diagnostic efficacy, that is, the sensitivity and specificity of the ddPCR assay compared with the synchronous blood culture. Secondary outcomes include the mortality rates and the mean Sequential Organ Failure Assessment (SOFA) score at follow-up time points, the length of stay in the hospital, the time to directed antimicrobial therapy, duration of broad-spectrum antibiotic use, and the EQ-5D-5L score on day 90. DISCUSSION: It is the first multicenter pragmatic RCT to explore the diagnostic efficacy and clinical impact of the ddPCR assay in patients with suspected sepsis, taking advantage of both RCT's ability to establish causality and the feasibility of pragmatic approaches in real-world studies (RWS). This trial will help us to get a comprehensive view of the assay's capacity for precise diagnosis and treatment of sepsis. It has the potential to monitor the pathogen load change and to guide the antimicrobial therapy, making a beneficial impact on the prognosis of sepsis patients. TRIAL REGISTRATION: ClinicalTrial.gov, NCT05190861. Registered January 13, 2022-'Retrospectively registered', https://clinicaltrials.gov/ct2/show/NCT05190861 .

Prognostic value of poly-microorganisms detected by droplet digital PCR and pathogen load kinetics in sepsis patients: a multi-center prospective cohort study.

This study aimed to investigate the prognostic value of a novel droplet digital polymerase chain reaction (DDPCR) assay in sepsis patients. In this prospective cohort study, univariable and multivariable Cox regressions were used to assess risk factors for 28-day mortality. We also monitored pathogen load together with clinical indicators in a subgroup of the cohort. A total of 107 sepsis patients with positive baseline DDPCR results were included. Detection of poly-microorganisms [adjusted hazard ratio (HR) = 3.19; 95% confidence interval (CI) = 1.34-7.62; P = 0.009], high Charlson Comorbidity Index (CCI) score (adjusted HR = 1.14; 95% CI = 1.01-1.29; P = 0.041), and Sequential Organ Failure Assessment (SOFA) score (adjusted HR = 1.18; 95% CI = 1.05-1.32; P = 0.005) at baseline were independent risk factors for 28-day mortality while initial pathogen load was not associated (adjusted HR = 1.17; 95% CI = 0.82-1.66; P = 0.385). Among 63 patients with serial DDPCR results, an increase in pathogen load at days 6-8 compared to baseline was a risk factor for 28-day mortality (P = 0.008). Also, pathogen load kinetics were significantly different between day-28 survivors and nonsurvivors (P = 0.022), with a decline overtime only in survivors and an increase from days 3 and 4 to days 6-8 in nonsurvivors. Using DDPCR technique, we found that poly-microorganisms detected and increased pathogen load a week after sepsis diagnosis were associated with poor prognosis.IMPORTANCEThis prospective study was initiated to explore the prognostic implications of a novel multiplex PCR assay in sepsis. Notably, our study was the largest cohort of sepsis with droplet digital polymerase chain reaction pathogen monitoring to date, allowing for a comprehensive evaluation of the prognostic significance of both pathogen species and load. We found that detection of poly-microorganisms was an independent risk factors for 28-day mortality. Also, pathogen load increase 1 week after sepsis diagnosis was a risk factor for 28-day mortality, and differential pathogen load kinetics were identified between day-28 survivors and nonsurvivors. Overall, this study demonstrated that pathogen species and load were highly correlated with sepsis prognosis. Patients exhibiting conditions mentioned above face a more adverse prognosis, suggesting the potential need for an escalation of antimicrobial therapy.Registered at ClinicalTrials.gov (NCT05190861).

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.

Genomic tracking and precise control of Klebsiella pneumoniae transmission in a newly established hospital: a prospective molecular epidemiological study.

OBJECTIVES: Carbapenem-resistant Klebsiella pneumoniae (CRKP) pose an emerging clinical threat. We investigated its introduction and transmission in a new hospital, evaluating the effect of whole-genome sequencing (WGS) as an infection control measure. METHODS: Based on WGS of identified K. pneumoniae (Kpn) strains, a prospective molecular epidemiological study of nosocomial transmission of CRKP in a newly established Chinese hospital was conducted. RESULTS: Between September 2018 and August 2020, 206 Kpn strains were isolated, including 180 CRKP, from 152 patients. The first imported and nosocomial transmission cases were recorded in December 2018 and April 2019, respectively. Overall, 22 nosocomial transmission clusters involving 85 patients were identified, among which 5 were large-size clusters comprising 5-18 patients. Index cases of the large-size clusters were more likely associated with lower Glasgow Coma Scale scores than those of small-size clusters. Furthermore, results of multivariable logistic regression indicated that Kpn tended to transmit more among patients in the ICU [adjusted odds ratio (aOR) = 4.96, 95% confidence interval (CI) 1.97-13.47] and those infected with a ST11 strain (aOR = 8.04, 95% CI 2.51-29.53) or tetracycline-resistant strains (aOR = 17.63, 95% CI 6.32-57.32). However, transmission was less likely in strains bearing the rmpA gene (aOR = 0.12, 95% CI 0.03-0.37). The rate of nosocomial CRKP cases decreased by 2.25 with the intervention of WGS-based infection control. CONCLUSIONS: Kpn transmission in the newly established hospital originated from several imported cases. Rates of nosocomial CRKP infection were reduced considerably through precise infection control measures.

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.

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.

The direct application of plasma droplet digital PCR in the ultra-early pathogen detection and warning during sepsis: Case reports.

BACKGROUND: Sepsis is a severe and acute disease associated with high mortality, for those who survive, long-term morbidity. In-time detection of pathogen for proper and optimized treatment in the early stage of sepsis is crucial. METHODS: We performed droplet digital PCR (ddPCR) on two cases of sepsis. The clinical information, laboratory test results, and the therapeutic regimen was detailed recorded. RESULTS: ddPCR showed its clinical value of sepsis ultra-early diagnosis and the guidance of medication in both cases we provided. CONCLUSIONS: ddPCR has potential for rapid and precise diagnose in ultra-early stage of sepsis for its high sensitivity and short turn-around-time, thus benefiting in optimal and timely treatment of sepsis.

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.

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.

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.