Improving the diagnosis of active tuberculosis: a novel approach using magnetic particle-based chemiluminescence LAM assay.

OBJECTIVES: Tuberculosis (TB) is a significant global health concern, given its high rates of morbidity and mortality. The diagnosis using urine lipoarabinomannan (LAM) primarily benefits HIV co-infected TB patients with low CD4 counts. The focus of this study was to develop an ultra-sensitive LAM assay intended for diagnosing tuberculosis across a wider spectrum of TB patients. DESIGN & METHODS: To heighten the sensitivity of the LAM assay, we employed high-affinity rabbit monoclonal antibodies and selected a highly sensitive chemiluminescence LAM assay (CLIA-LAM) for development. The clinical diagnostic criteria for active TB (ATB) were used as a control. A two-step sample collection process was implemented, with the cutoff determined initially through a ROC curve. Subsequently, additional clinical samples were utilized for the validation of the assay. RESULTS: In the assay validation phase, a total of 87 confirmed active TB patients, 19 latent TB infection (LTBI) patients, and 104 healthy control samples were included. Applying a cutoff of 1.043 (pg/mL), the CLIA-LAM assay demonstrated a sensitivity of 55.2% [95%CI (44.13%~65.85%)], and a specificity of 100% [95%CI (96.52%~100.00%)], validated against clinical diagnostic results using the Mann-Whitney U test. Among 11 hematogenous disseminated TB patients, the positive rate was 81.8%. Importantly, the CLIA-LAM assay consistently yielded negative results in the 19 LTBI patients. CONCLUSION: Overall, the combination of high-affinity antibodies and the CLIA method significantly improved the sensitivity and specificity of the LAM assay. It can be used for the diagnosis of active TB, particularly hematogenous disseminated TB.

Breakthrough of chemiluminescence-based LAM urine test beyond HIV-positive individuals: Clinical diagnostic value of pulmonary tuberculosis in the general population.

To investigate the diagnostic value of a novel high-sensitivity urine lipoarabinomannan (LAM) test (chemiluminescence-based) for active tuberculosis in the general population. A retrospective study was conducted on 250 clinical suspected tuberculosis patients who were HIV-negative and visited the Fourth People's Hospital of Foshan from January 2022 to December 2022. Among them, there were 135 cases of pulmonary tuberculosis, 34 cases of extrapulmonary tuberculosis, and 81 cases of non-tuberculosis. Urine samples were collected for LAM antigen detection before treatment, and laboratory data of sputum smear acid-fast staining (smear method), sputum culture, and GeneXpert method were collected. Using clinical diagnosis as the reference standard, the diagnostic efficacy of 4 methods for detecting active tuberculosis was evaluated. For the 135 cases of pulmonary tuberculosis, the sensitivity of sputum smears, sputm culture, sputm GeneXpert method, and urine LAM were 29.6% (40/135), 45.9% (62/135), 59.3% (80/135), and 51.9% (70/135), respectively. The combination of LAM + GeneXpert and LAM + culture had the highest sensitivity for detecting active pulmonary tuberculosis, which were 71.0% and 78.2%, respectively. For the detection of sputum culture-negative pulmonary tuberculosis, the positive rates of smear, GeneXpert, and LAM were 0.0% (0/73), 53.4% (39/73), and 52.1% (38/73), respectively. LAM + smear and LAM + Genexpert could detect 52.1% and 68.5% of sputum culture-negative patients, respectively. The high-sensitivity urine LAM test holds promise for tuberculosis diagnosis in the general population. It demonstrates high-sensitivity, enabling the detection of sputum culture-negative pulmonary tuberculosis patients. Furthermore, when combined with existing methods, it can enhance the overall detection rate.

Insufficient epitope-specific T cell clones are responsible for impaired cellular immunity to inactivated SARS-CoV-2 vaccine in older adults.

Aging is a critical risk factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine efficacy. The immune responses to inactivated vaccine for older adults, and the underlying mechanisms of potential differences to young adults, are still unclear. Here we show that neutralizing antibody production by older adults took a longer time to reach similar levels in young adults after inactivated SARS-CoV-2 vaccination. We screened SARS-CoV-2 variant strains for epitopes that stimulate specific CD8 T cell response, and older adults exhibited weaker CD8 T-cell-mediated responses to these epitopes. Comparison of lymphocyte transcriptomes from pre-vaccinated and post-vaccinated donors suggested that the older adults had impaired antigen processing and presentation capability. Single-cell sequencing revealed that older adults had less T cell clone expansion specific to SARS-CoV-2, likely due to inadequate immune receptor repertoire size and diversity. Our study provides mechanistic insights for weaker response to inactivated vaccine by older adults and suggests the need for further vaccination optimization for the old population.

A new pattern of citrullinated peptides improves the sensitivity for diagnosing rheumatoid arthritis.

OBJECTIVES: Anti-citrullinated protein antibody (ACPA) is found almost exclusively in patients with rheumatoid arthritis (RA). Commercial cyclic citrullinated peptide (CCP) assays that target ACPAs yield specificities as high as 95% for RA diagnoses, but their sensitivities only reach 50-70%. To improve the sensitivity of the CCP assay, a new pattern of citrullinated peptide was identified and named the MCSM (multiple citrulline-similar motif). DESIGN & METHODS: The MCSM comprised a citrulline core surrounded by citrulline-similar amino acids. A series of peptides with or without the MCSM was synthesized to evaluate the function of the citrulline core and citrulline-similar amino acids. These peptides were used in the enzyme-linked immunosorbent assay to compare samples from 94 RA patients, 117 non-RA patients and 116 healthy subjects. Additionally, the MCSM assay was compared with a commercial CCP assay. RESULTS: When the cutoff value was set at 0.274, the sensitivity and specificity of the MCSM assay were 79.6% and 96.6%, respectively. When one citrulline was substituted in the citrulline core, the sensitivity of the assay decreased from 79.6% to 61%. If all three citrulline-similar amino acids were substituted in the backbone, the sensitivity of the MCSM assay decreased from 79.6% to 58.5%. The coincidence rate of the MCSM assay to the commercial CCP assay was 97.6%. CONCLUSIONS: The citrulline core and citrulline-similar amino acids are crucial components of the MCSM pattern. This new MCSM assay could be used to diagnose RA.

Citrullinated Antigens with Multiple Citrulline Similar Motif in the Diagnosis of Rheumatoid Arthritis: A Preliminary Single-Center Study.

The presence of anti-citrullinated protein antibodies (ACPAs) in the serum is one of the immunological features of rheumatoid arthritis (RA). Anti-cyclic citrullinated peptide (CCP) assay has been widely used in clinic for the diagnosis of RA. However, up to 40% of RA patients are anti-CCP negative and the diagnostic sensitivity in this population needs to be improved for better clinical management. In this study, peptides with Multiple Citrulline Similar Motif (MCSM) were synthesized and a new ELISA system, which we called RA_CP, was developed to detect citrullinated antigens with MCSM present in the serum. 106 RA,48 other arthritis patients and 41 sex- and age-matched healthy controls (HCs) were included in this study. Patients with RA have a significantly higher amount of citrullinated antigens with MCSM than other arthritis patients and HCs. RA patients with positive anti-CCP are also MCSM positive, whereas 75% anti-CCP negative patients are positive for MCSM. The diagnostic sensitivity for anti-CCP and MCSM was 81.1% and 95.3%, while the specificity was 100% and 94.4%, respectively. ROC curve analyses showed that the area under the curve (AUC) values were 0.906 (95% CI: 0.860-0.951) for anti-CCP and 0.948 (95% CI: 0.912-0.985) for MCSM while the combination of MCSM and anti-CCP test has the highest AUC (0.971, 95% CI: 0.946-0.996). Our results suggest that detection of citrullinated antigens with MCSM has improved sensitivity compared with anti-CCP assay and could serve as a biomarker in diagnosis of RA patients.

The next-generation Hybrid Capture High-Risk HPV DNA assay on a fully automated platform.

BACKGROUND: A next-generation diagnostic system has been developed at QIAGEN. The QIAensemble system consists of an analytical subsystem (JE2000) that utilizes a re-engineered Hybrid Capture chemistry (NextGen) to maintain the high level of clinical sensitivity established by the digene High-Risk HPV DNA Test (HC2), while creating improved analytical specificity as shown both in plasmid-based analyses and in processing of clinical specimens. STUDY DESIGN: Limit-of-detection and cross-reactivity experiments were performed using plasmid DNA constructs containing multiple high-risk (HR) and low-risk (LR) HPV types. Cervical specimens collected into a novel specimen collection medium, DCM, were used to measure stability of specimens, as well as analytical specificity. Signal carryover, instrument precision, and specimen reproducibility were measured on the prototype JE2000 system using the automated NextGen assay. RESULTS: The Limit of Detection (LOD) is <1000 copies of HPV 16 plasmid in the automated assay. No cross-reactivity (signal above cutoff) was detected on the automated system from any of 13 LR types tested at 10(7) copies per assay. Within-plate, plate-to-plate, and day-to-day performance in the prototype system yielded a CV of 20%. No indication of target carryover was found when samples containing up to 10(9) copies/ml of HPV DNA type 16 were processed on the JE2000 instrument. In an agreement study with HC2, 1038 donor cervical specimens were tested in both the manual NextGen assay and HC2 to evaluate agreement between the two tests. After eliminating discrepant specimens that were adjudicated by HR-HPV genotyping, the adjudicated positive agreement was 98.5% (95% CI: 94.6, 99.6). CONCLUSIONS: The JE2000 prototype system automates NextGen assay processing, yielding accurate, reproducible, and highly specific results with both plasmid analytical model tests and cervical specimens collected in DCM. The final system will process more than 2000 specimens in an 8-hour shift, with fully continuous loading.

Proteomic profiling identifies cyclooxygenase-2-independent global proteomic changes by celecoxib in colorectal cancer cells.

Celecoxib, a selective inhibitor of the enzyme cyclooxygenase-2 (COX-2), has been shown to be a promising chemoprevention agent. The chemopreventive efficacy of celecoxib is believed to be a consequence of its COX-2-dependent and COX-2-independent effects on a variety of cellular processes including proliferation, apoptosis, angiogenesis, and immunosurveillance. In an attempt to identify proteomic markers modulated by celecoxib that are independent of its inhibitory effect on COX-2, the colorectal cancer cell line HCT-116, a nonexpresser of COX-2, was treated with celecoxib. We used the powerful, state-of-the-art two-dimensional difference gel electrophoresis technology coupled with mass spectrometric sequencing to compare global proteomic profiles of HCT-116 cells before and after treatment with celecoxib. Among the differentially expressed proteins identified following celecoxib treatment were proteins involved in diverse cellular functions including glycolysis, protein biosynthesis, DNA synthesis, mRNA processing, protein folding, phosphorylation, redox regulation, and molecular chaperon activities. Our study presents a comprehensive analysis of large-scale celecoxib-modulated proteomic alterations, at least some of which may be mechanistically related to the COX-2-independent chemopreventive effect of celecoxib.

Multimerization via its myosin domain facilitates nuclear localization and inhibition of core binding factor (CBF) activities by the CBFbeta-smooth muscle myosin heavy chain myeloid leukemia oncoprotein.

In CBFbeta-SMMHC, core binding factor beta (CBFbeta) is fused to the alpha-helical rod domain of smooth muscle myosin heavy chain (SMMHC). We generated Ba/F3 hematopoietic cells expressing a CBFbeta-SMMHC variant lacking 28 amino acids homologous to the assembly competence domain (ACD) required for multimerization of skeletal muscle myosin. CBFbeta-SMMHC(DeltaACD) multimerized less effectively than either wild-type protein or a variant lacking a different 28-residue segment. In contrast to the control proteins, the DeltaACD mutant did not inhibit CBF DNA binding, AML1-mediated reporter activation, or G(1) to S cell cycle progression, the last being dependent upon activation of CBF-regulated genes. We also linked the CBFbeta domain to 149 or 83 C-terminal CBFbeta-SMMHC residues, retaining 86 or 20 amino acids N-terminal to the ACD. CBFbeta-SMMHC(149C) multimerized and slowed Ba/F3 proliferation, whereas CBFbeta-SMMHC(83C) did not. The majority of CBFbeta-SMMHC and CBFbeta-SMMHC(149C) was detected in the nucleus, whereas the DeltaACD and 83C variants were predominantly cytoplasmic, indicating that multimerization facilitates nuclear retention of CBFbeta-SMMHC. When linked to the simian virus 40 nuclear localization signal (NLS), a significant fraction of CBFbeta-SMMHC(DeltaACD) entered the nucleus but only mildly inhibited CBF activities. As NLS-CBFbeta-SMMHC(83C) remained cytoplasmic, we directed the ACD to CBF target genes by linking it to the AML1 DNA binding domain or to full-length AML1. These AML1-ACD fusion proteins did not affect Ba/F3 proliferation, in contrast to AML1-ETO, which markedly slowed G(1) to S progression dependent upon the integrity of its DNA-binding domain. Thus, the ACD facilitates inhibition of CBF by mediating multimerization of CBFbeta-SMMHC in the nucleus. Therapeutics targeting the ACD may be effective in acute myeloid leukemia cases associated with CBFbeta-SMMHC expression.