Impact of DHCWs' Safety Perception on Vaccine Acceptance and Adoption of Risk Mitigation Strategies.

OBJECTIVES: To estimate the association between safety perception on vaccine acceptance and adoptions of risk mitigation strategies among dental health care workers (DHCWs). METHODS: A survey was emailed to DHCWs in the New Jersey area from December 2020 to January 2021. Perceived safety from regular SARS-CoV-2 testing of self, coworkers, and patients and its association with vaccine hesitancy and risk mitigation were ascertained. Risk Mitigation Strategy (RiMS) scores were computed from groupings of office measures: 1) physical distancing (reduced occupancy, traffic flow, donning of masks, minimal room crowding), 2) personal protective equipment (fitted for N95; donning N95 masks; use of face shields; coverings for head, body, and feet), and 3) environmental disinfection (suction, air filtration, ultraviolet, surface wiping). RESULTS: SARS-CoV-2 testing of dental professionals, coworkers, and patients were perceived to provide safety at 49%, 55%, and 68%, respectively. While dentists were least likely to feel safe with regular self-testing for SARS-CoV-2 (P < 0.001) as compared with hygienists and assistants, they were more willing than hygienists (P = 0.004; odds ratio, 1.79 [95% CI, 1.21 to 2.66]) and assistants (P < 0.001; odds ratio, 3.32 [95% CI, 1.93 to 5.71]) to receive the vaccine. RiMS scores ranged from 0 to 19 for 467 participants (mean [SD], 10.9 [2.9]). RiMS scores did not significantly differ among groups of DHCWs; however, mean RiMS scores were higher among those who received or planned to receive the COVID-19 vaccine than those with who did not (P = 0.004). DHCWs who felt safer with regular testing had greater RiMS scores than those who did not (11.0 vs. 10.3, P = 0.01). CONCLUSIONS: Understanding DHCWs' perception of risk and safety is crucial, as it likely influences attitudes toward testing and implementation of office risk mitigation policies. Clinical studies that correlate risk perception and RiMS with SARS-CoV-2 testing are needed to demonstrate the effectiveness of RiMS in dental care settings. KNOWLEDGE TRANSFER STATEMENT: Educators, clinicians, and policy makers can use the results of this study when improving attitudes toward testing and implementation of risk mitigation policies within dental offices, for current and future pandemics.

Antibody markers of incident tuberculosis among HIV-infected adults in the USA: a historical prospective study.

OBJECTIVE: To assess whether serum levels of antibodies against Mycobacterium tuberculosis antigens increase before diagnosis of active tuberculosis (TB). DESIGN: Serial serum samples were obtained from 30 human immunodeficiency virus (HIV) co-infected individuals who developed active TB during a multicenter prospective study on pulmonary complications of HIV/AIDS conducted among >1300 subjects in the USA in the 1980s. Multiple serum samples from 47 matched control individuals who did not develop TB in the same study were also tested. Immunoglobulin G (IgG) antibodies to 10 M. tuberculosis proteins were detected by enzyme-linked immunosorbent assay (ELISA), and data were analyzed by descriptive and inferential statistical techniques to assess patterns, trends and differences in antibody levels relative to time from TB diagnosis. RESULTS: Antibodies to five antigens (ESAT-6, 38 kDa Ag, 16 kDa Ag, malate synthase and MTSA-10/CFP-10), but not to five other antigens (Rv2626c, ferredoxin A, glutamine synthetase, alanine dehydrogenase and Ag85) increased before diagnosis of TB relative to control levels. The earliest increase in the TB group was detected for MTSA-10/CFP-10 (24-30 months pre-diagnosis). CONCLUSIONS: Levels of serum antibodies to particular proteins of M. tuberculosis increase before microbiological and clinical symptoms of active TB. The use of antibody biomarkers for prognostic purposes should therefore be feasible.

Serological markers of pulmonary tuberculosis and of response to anti-tuberculosis treatment in a patient population in Guinea.

The aim of the study was to evaluate serological correlates of active tuberculosis and of response to antituberculosis treatment in a cohort of HIV-negative patients with pulmonary tuberculosis studied at diagnosis and during treatment at the Service de Pneumo-Phtisiologie, Centre Hospitalier-Universitaire Ignace Deen, Conakry, Republic of Guinea. Two similar cohorts of HIV-negative healthy households of patients and healthy community controls were included in the study. Plasma samples were obtained from 168 untreated tuberculosis patients, 167 healthy household controls, and 168 healthy community controls. Serial plasma samples were also obtained from the tuberculosis patients at 2 and 8 months after initiation of chemotherapy. IgG antibody levels were measured by an enzyme-linked immunosorbent assay (ELISA) using ten purified M. tuberculosis antigens. ELISA results were analysed by comparing geometric means of data. Of the ten antigens tested, five (14kDa Ag, 19kDa Ag, AlaDH, MS, and MPT83) elicited similar antibody responses in untreated TB patients and controls. In contrast, levels of three antibodies (ESAT-6, LAM, and 38kDa Ag) were higher in untreated TB patients than in household or community controls (p<0.0001). Levels were higher in untreated patients than in community controls also for the anti-Rv2626c antibody (p = 0.0001) and, at a lower significance level, for the anti-FdxA antibody (p<0.025). Antibody levels against ESAT-6 and Rv2626c decreased during therapy, while antibody levels to the 38 kDa antigen and LAM increased during therapy; FdxA antibody levels did not vary with treatment. Neither severity of presentation nor chest X-ray patterns affected levels of these antibodies before treatment. In contrast, after the 8-month therapeutic course, patients who presented with moderate/severe disease had higher levels of anti-ESAT-6, anti-FdxA, and anti-38kDa antibodies than those of patients with mild disease onset. Patients with bilateral lung lesions had significantly higher anti-38kDa and anti-LAM levels, both at diagnosis and after 8-month treatment, than patients with lesions involving only one lung. Antibodies to alanine dehydrogenase and malate synthetase measured at initiation of treatment were higher in tuberculosis patients who subsequently failed therapy than in those who were cured. The main conclusions of the study are: a) plasma levels of antibodies to a number of M. tuberculosis represent serological correlates of active disease; b) these correlates are affected in an antigen-specific fashion by anti-tuberculosis treatment; c) particular serological markers may be predictive of treatment outcome.

Integration of microscopy and serodiagnostic tests to screen for active tuberculosis.

SETTING: University of California San Diego Medical Center, USA. OBJECTIVE: To create a simple screening strategy for tuberculosis (TB) that includes antibody detection assays to improve the accuracy of microscopic examination of sputum for acid-fast bacilli (AFB smear). METHODS: Serum samples were obtained from 190 patients suspected of having active TB. TB diagnosis was established by Mycobacterium tuberculosis culture. HIV status was determined by commercial serologic tests. IgG antibody levels were measured by ELISA using purified M. tuberculosis antigens. Data from 130 randomly selected patients were used to develop a screening strategy; data from the remaining 60 patients were used for validation. RESULTS: AFB smear had 70% sensitivity and 88% specificity. In algorithms integrating single or multi-antigen ELISA with AFB smear and HIV results, the sensitivity improved over each test alone. The algorithm that included a four-antigen ELISA (38 kDa antigen, lipoarabinomannan, MPT-64 and glutamine synthase) had a sensitivity of 93% and a specificity of 76%. Compared to AFB smear, the sensitivity of the algorithm was significantly higher, while the specificity was not statistically different. CONCLUSION: This study demonstrates that a screening strategy can be created by integrating multi-antigen ELISA with AFB smear and HIV testing.

Analyzing Mycobacterium tuberculosis proteomes for candidate vaccine epitopes.

Secreted antigens of Mycobacterium tuberculosis (Mtb) induce strong T cell responses and interferon-gamma (IFN-gamma) secretion, both of which are integral in the defense against Mtb. We used web-based tools (SignaIP and Prosite) to identify putative secreted proteins from Mtb genomes CDC 1551 and H37Rv. We then used EpiMatrix, a proprietary pattern-matching algorithm, to do a preliminary analysis of these proteins for regions that contained a high number of class II MHC binding motif matches. The use of bioinformatics tools reduced the number of potential epitopes to be screened to 5% of the 1.3 million overlapping peptides. Peripheral blood mononuclear cells (PBMC) were obtained from healthy, asymptomatic tuberculin skin test-positive donors. Of the 17 highest-ranking peptide candidates that could be synthesized for this preliminary in vitro evaluation, 15 (88%) stimulated IFN-gamma response, and eight (47%) stimulated lymphocyte proliferation in vitro. IFN-gamma ELISpot assays were therefore a more sensitive test for T cell response to these peptides than were proliferation assays. One highly promiscuous epitope (MT2281-26-J, WRRRPLSSALLSFGLLLGGLPL) induced IFN-gamma secretion in PBMC from 11 of 25 Mtb immune subjects (44%). Overall, 15 epitopes, and MT2281-26-J in particular, are candidates for inclusion in a multi-epitope TB vaccine. These findings support the systematic application of bioinformatics tools to whole genomes when used in combination with in vitro methods for screening and confirming epitopes.

Immunological characterization of novel secreted antigens of Mycobacterium tuberculosis.

Proteins secreted by Mycobacterium tuberculosis are targets of host immune responses and as such are investigated for vaccine and immunodiagnostics development. Computer-driven searches of the M. tuberculosis H37Rv genome had previously identified 45 novel secreted proteins. Here, we report the characterization of these antigens in terms of specificity for the M. tuberculosis complex and the ability to induce human immune responses. BLAST homology searches and Southern hybridization identified 10 genes that were either specific for the M. tuberculosis complex or found in only two nontuberculous mycobacterial species of minor medical significance. Selected recombinant proteins were purified from Escherichia coli cells and tested for the ability to elicit antibody responses in tuberculosis patients. Reactivity of the serum panel was ' 36% with at least one of five novel proteins (Rv0203, Rv0603, Rv1271c, Rv1804c and Rv2253), 56% with the 38 kDa lipoprotein, a M. tuberculosis antigen known to be highly seroreactive, and 68% with a combination of Rv0203, Rv1271c and the 38 kDa antigen. Thus, at least five novel secreted proteins induce antibody responses during active disease; some of these proteins may increase the sensitivity of serological assays based on the 38 kDa antigen.

Tuberculosis immunology in children: diagnostic and therapeutic challenges and opportunities.

Tuberculosis (TB) is one of the most important causes of infectious morbidity and mortality worldwide. Young children are more likely to develop severe disease from the causative agent Mycobacterium tuberculosis. These clinical observations likely reflect fundamental differences in the immune systems of young children and adults. Essential to effective TB immunity are functioning macrophages, dendritic cells, strong Th1-type T-cell immunity and a relative absence of Th2-type T-cell immunity. Critical differences between adults and children relevant to TB immunity include deficiencies in macrophage and dendritic cell function, deficiencies in the development of Th1-type T-cells in response to pathogens, and the propensity for infants and young children to develop Th2-type CD4+ T-cells in response to immunogens. In this article, knowledge about the requisite components of protective immunity, differences between the immune systems of children and adults relevant to pediatric tuberculosis, M. tuberculosis-specific T-cell immunity in children, and potential application to immunodiagnostics and vaccine development will be reviewed.

Recognition of ESAT-6 sequences by antibodies in sera of tuberculous nonhuman primates.

Previous work in our laboratory showed that the ESAT-6 protein of Mycobacterium tuberculosis and Mycobacterium bovis induces strong antibody responses in a large proportion ( approximately 90%) of experimentally or naturally infected nonhuman primates. Here, the antibody response to ESAT-6 in tuberculous monkeys was characterized at the epitope level by measuring antibodies to overlapping, synthetic peptides spanning the ESAT-6 sequence. The antibody response against the COOH-terminal portion of the protein was the strongest in both experimentally and naturally infected animals. Moreover, these antibodies became detectable the earliest during experimental infection, suggesting an ordered expansion of ESAT-6-specific B-cell clones in the course of infection. The data support use of synthetic peptides in lieu of the full-length ESAT-6 protein in diagnostic antibody detection assays.

Factors associated with humoral response to ESAT-6, 38 kDa and 14 kDa in patients with a spectrum of tuberculosis.

SETTING: Tertiary care chest hospital in Montreal, Canada, where the average annual incidence of TB is 10/100,000 population. OBJECTIVES: To evaluate the clinical correlates of humoral response to three Mycobacterium tuberculosis antigens. METHODS: Humoral response to three M. tuberculosis antigens, 38 kDa, 14 kDa and ESAT-6, was measured with ELISA in patients with a spectrum of TB-related conditions. The association of positive tests for each antigen, defined with receiver operator characteristics (ROC) analysis, and patient characteristics was assessed in multivariate regression. RESULTS: A total of 383 patients underwent serologic testing. In multivariate analysis, humoral response to 38 kDa was associated with active disease, response to 14 kDa was associated with inactive TB and female sex, and response to ESAT-6 with inactive TB, female sex, prior contact with TB, and recent arrival in Canada from high prevalence countries. CONCLUSIONS: Response to the 38 kDa antigen was associated with current active disease, and was very different from response to the 14 kDa and ESAT-6 antigens. These latter two antigens were associated with risk factors for future active, but not current disease, suggesting that they might be useful to identify persons with higher risk of reactivation of latent TB.

Immunological characterization of antigens encoded by the RD1 region of the Mycobacterium tuberculosis genome.

Development of immunoassays specific for the diagnosis of tuberculosis requires antigens unique to Mycobacterium tuberculosis. In a search for such antigens we tested six proteins encoded by RD1, a region present in M. tuberculosis and virulent M. bovis genomes but missing from the DNA of all substrains of M. bovis Bacillus Calmette-Guerin (BCG). The six proteins (Rv3871, Rv3872, Rv3873, MTSA-10, ESAT-6 and Rv3878) were purified to near-homogeneity from recombinant Escherichia coli. When tested for the ability to elicit antibody responses and delayed type hypersensitivity in tuberculous guinea pigs, only two of six antigens, ESAT-6 and MTSA-10, elicited strong skin reactions, while vigorous antibody responses were observed to all six proteins. When antibody responses to RD1 antigens were evaluated in sera from patients having pulmonary tuberculosis and from control subjects (patients having mycobacterioses other than tuberculosis, and healthy persons), a sizeable proportion (25%) of tuberculosis patients but none of the control subjects, had antibodies against MTSA-10 and/or ESAT-6. We conclude that MTSA-10 and ESAT-6 are promising candidates for immunodiagnostic assays specific for tuberculosis.

Characterization of the secreted MPT53 antigen of Mycobacterium tuberculosis.

MPT53 is a secreted protein of Mycobacterium tuberculosis. Southern transfer and hybridization showed mpt53 to be conserved in the M. tuberculosis complex and to have homology with DNA from Mycobacterium avium and other nontuberculous mycobacteria. However, anti-MPT53 polyclonal antibodies detected no antigen in the culture filtrates of M. avium and other nontuberculous mycobacteria. MPT53 of M. tuberculosis induced strong, tuberculosis-specific antibody responses in guinea pigs but induced no delayed-type hypersensitivity. Involvement in immune responses during human tuberculosis was very modest.

Combinatorial use of antibodies to secreted mycobacterial proteins in a host immune system-independent test for tuberculosis.

Laboratory diagnosis of tuberculosis is often difficult. Immunodetection of circulating Mycobacterium tuberculosis proteins shed during active infection would not depend on an intact host immune response and could take advantage of the speed and low costs afforded by antibody-based assays. We previously showed that patients with active tuberculosis had increased levels of circulating antigen 85 (Ag85) proteins independent of their tuberculin skin test status (S. I. Bentley-Hibbert, X. Quan, T. Newman, K. Huygen, and H. P. Godfrey, Infect. Immun. 67:581-588, 1999). To extend these observations to a Mycobacterium bovis BCG-vaccinated population and to another secreted mycobacterial protein, Ag85 and PstS-1 (protein antigen B, p38 antigen) were quantified in sera from 97 Chilean tuberculosis patients and healthy controls (many of whom had received BCG as children) using dot immunobinding, mouse monoclonal anti-BCG Ag85 complex antibody, and chicken antipeptide antibodies reactive with M. tuberculosis Ag85B and PstS-1. The latter antibodies had been raised to peptide-derived immunogens expressed on a novel proprietary protein carrier in Escherichia coli. Median serum Ag85 levels measured by using either anti-Ag85 antibody were significantly higher in patients with active tuberculosis than in healthy controls (P, <0.001 to 0.01); the median serum PstS-1 levels were similar in patients and controls. The sensitivity of significantly elevated circulating Ag85 levels in patients with pulmonary tuberculosis measured by anti-Ag85 complex or anti-Ag85B antibodies was 60 and 55%, respectively, but increased to 77% when results obtained with both anti-Ag85 antibodies were considered jointly (P < 0.02). The corresponding specificities for individual and joint consideration were 95, 85, and 80%, respectively. These results indicate that elevated Ag85 levels can be detected in patients with active tuberculosis even after BCG vaccination and suggest that combinatorial use of antibodies directed at different epitopes of this protein could provide a viable strategy for developing new host immune response-independent diagnostic tests for tuberculosis.

Accuracy and utility of commercially available amplification and serologic tests for the diagnosis of minimal pulmonary tuberculosis.

Diagnosis of patients with minimal active tuberculosis (TB) is difficult, as there is no single test with high sensitivity and specificity. The yield and clinical utility of a combination of diagnostic tests were prospectively studied among 500 consecutive patients referred for sputum induction for diagnosis of possible active TB. Patients underwent sputum induction, chest X-ray, tuberculin testing, and had blood drawn for serologic testing (Detect-TB test; Biochem ImmunoSystems). Sputum was examined with fluorescent microscopy and PCR (Amplicor MTB-Roche) and cultured for mycobacteria using liquid (BACTEC) and solid media. For the diagnosis of the 60 cases of active TB, sensitivity and specificity, respectively, of the following diagnostic tests were mycobacterial culture, 73% and 100%; PCR, 42% and 100%; chest X-ray, 67-77% and 66-76%; tuberculin testing, 94% and 20%; and serology, 33% and 87%. After consideration of PCR and radiographic and clinical characteristics, a positive serologic test was independantly associated with diagnosis of active disease (adjusted odds of disease if positive, 2.6; 95% confidence limits, 1.1,6.1). No currently available test has sensitivity and specificity high enough for the accurate diagnosis of minimal pulmonary TB. Utilization of a combination of tests, together with consideration of key clinical characteristics, could improve diagnostic accuracy.

A multi-antigen print immunoassay for the development of serological diagnosis of infectious diseases.

Serological diagnosis of infectious diseases that generate a highly heterogeneous antibody repertoire, such as tuberculosis, requires tests based on cocktails of antigens. We describe a new method called multi-antigen print immunoassay (MAPIA) for cocktail-based serological diagnosis. The assay entails the application of antigen to nitrocellulose membranes by micro-aerosolization (printing), followed by antibody detection using standard chromogenic immunodevelopment. Cocktails of protein antigens of Mycobacterium tuberculosis tested by MAPIA were found to maintain the serological activity of each of their components. In contrast, the same cocktails tested by enzyme-linked immunosorbent assay (ELISA) had a serological activity that was lower than the sum of the activities of their components. Consequently, cocktail-based MAPIA attained the diagnostic sensitivity expected on the basis of single antigen results, while a significant loss of diagnostic sensitivity was observed with cocktail-based ELISA. Thus, the MAPIA format is superior to conventional ELISA for the serological diagnosis of infectious diseases characterized by heterogeneous antibody responses.

Immunologic diagnosis of tuberculosis.

Evaluation of new vaccines against tuberculosis requires diagnostic tools for accurately identifying asymptomatic individuals infected with Mycobacterium tuberculosis and persons with active tuberculosis. This article discusses limitations of current methods for the immunologic diagnosis of latent infection and active disease and presents novel approaches to developing skin tests and serodiagnostic assays based on "cocktails" of multiple antigens of M. tuberculosis.

Identification of secreted proteins of Mycobacterium tuberculosis by a bioinformatic approach.

Proteins secreted by Mycobacterium tuberculosis are usually targets of immune responses in the infected host. Here we describe a search for secreted proteins that combined the use of bioinformatics and phoA' fusion technology. The 3,924 proteins deduced from the M. tuberculosis genome were analyzed with several computer programs. We identified 52 proteins carrying an NH(2)-terminal secretory signal peptide but lacking additional membrane-anchoring moieties. Of these 52 proteins-the TM1 subgroup-only 7 had been previously reported to be secreted proteins. Our predictions were confirmed in 9 of 10 TM1 genes that were fused to Escherichia coli phoA', a marker of subcellular localization. These findings demonstrate that the systematic computer search described in this work identified secreted proteins of M. tuberculosis with high efficiency and 90% accuracy.

MTSA-10, the product of the Rv3874 gene of Mycobacterium tuberculosis, elicits tuberculosis-specific, delayed-type hypersensitivity in guinea pigs.

In a search for new skin test reagents specific for tuberculosis, we found that the antigen encoded by gene Rv3874 of Mycobacterium tuberculosis elicited delayed-type hypersensitivity in M. tuberculosis-infected guinea pigs but not in control animals immunized with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium avium. The antigen, which was named MTSA-10 (for M. tuberculosis-specific antigen 10), is a prime candidate for a component of a new tuberculin that will allow discrimination by a skin test of latent M. tuberculosis infection from vaccination with BCG or from sensitization with environmental, nontuberculous mycobacteria.

Humoral immune responses to multiple antigens of Mycobacterium tuberculosis in tuberculosis patients co-infected with the human immunodeficiency virus.

A panel of ten protein antigens of Mycobacterium tuberculosis was used to evaluate serum antibody responses to tuberculosis in patients co-infected with the human immunodeficiency virus (HIV) and in HIV-infected control individuals without tuberculosis. Most (70%) of the tuberculosis patients had serum reactivity to at least one antigen and maintained the diverse antibody repertoire previously observed in HIV-negative tuberculosis patients.

HSa of Staphylococcus aureus, a new member of the HU family of bacterial histone-like proteins.

Histone-like proteins of the HU family are small, sequence-independent DNA binding proteins that facilitate a variety of DNA transactions. Here we report isolation from cell-free extracts of Staphylococcus aureus of HSa, a new member of the HU family. The NH2-terminal amino acid sequence of HSa led to identification of the corresponding gene (hsa) using the genome sequence of S. aureus. HSa is 90 amino acids long (Mr = 9 620) and shares 64 to 80% identity with homologs found in the genus Bacillus.

Differential T cell responses to Mycobacterium tuberculosis ESAT6 in tuberculosis patients and healthy donors.

Vaccination against and diagnosis of tuberculosis are still insufficient. Proteins secreted by Mycobacterium tuberculosis induce strong immune responses in tuberculosis and constitute prime candidates for development of novel vaccines against tuberculosis as well as for immunodiagnostic assays. We investigated the role of the secreted proteins MPT63, MPT64 and ESAT6 from M. tuberculosis in healthy individuals and tuberculosis patients. None of the secreted proteins stimulated peripheral blood mononuclear cells from healthy donors. In contrast, CD4+ T cells from many tuberculosis patients were stimulated in an MHC class II-restricted fashion by ESAT6, but not by MPT63 or MPT64. T cell reactivities of tuberculosis patients were focused on the N-terminal region of ESAT6. The ESAT6 T cell epitopes were presented by different HLA-DR phenotypes. Cell cultures responding to either ESAT6 or synthetic peptides thereof showed mRNA transcripts for macrophage inflammatory protein (MIP)-1alpha, monocyte chemotactic protein (MCP)-1 or IL-8 and production of IFN-gamma and MIP-1alpha. Our results suggest that the secreted M. tuberculosis proteins MPT63, MPT64 or ESAT6 do not stimulate unprimed T cells, and that ESAT6 may be a potential candidate antigen for detection of clinical disease.