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BackgroundTuberculosis (TB) infection was responsible for an estimated 1.3 million deaths in 2017. Better diagnostic tools are urgently needed. We sought to determine whether accurate TB antigen detection in blood or urine has the potential to meet the WHO target product profiles for detection of active TB.Materials and methodsWe developed Electrochemiluminescence (ECL) immunoassays for Lipoarabinomannan (LAM) and ESAT-6 detection with detection limits in the pg/ml range and used them to compare the concentrations of the two antigens in the urine and serum of 81 HIV-negative and -positive individuals with presumptive TB enrolled across diverse geographic sites.ResultsLAM and ESAT-6 overall sensitivities in urine were 93% and 65% respectively. LAM and ESAT-6 overall sensitivities in serum were 55% and 46% respectively. Overall specificity was ≥97% in all assays. Sensitivities were higher in HIV-positive compared to HIV-negative patients for both antigens and both sample types, with signals roughly 10-fold higher on average in urine than in serum. The two antigens showed similar concentration ranges within the same sample type and correlated.ConclusionsLAM and ESAT-6 can be detected in the urine and serum of TB patients, regardless of the HIV status and further gains in clinical sensitivity may be achievable through assay and reagent optimization. Accuracy in urine was higher with current methods and has the potential to meet the WHO accuracy target if the findings can be transferred to a point-of-care TB test.

The only currently commercialized point-of-care assay for tuberculosis (TB) that measures lipoarabinomannan (LAM) in urine (Alere LF-LAM) has insufficient sensitivity. We evaluated the potential of 100 novel monoclonal antibody pairs targeting a variety of LAM epitopes on a sensitive electrochemiluminescence platform to improve the diagnostic accuracy. In the screening, many antibody pairs showed high reactivity to purified LAM but performed poorly at detecting urinary LAM in clinical samples, suggesting differences in antigen structure and immunoreactivity of the different LAM sources. The 12 best antibody pairs from the screening were tested in a retrospective case-control study with urine samples from 75 adults with presumptive TB. The best antibody pair reached femtomolar analytical sensitivity for LAM detection and an overall clinical sensitivity of 93% (confidence interval [CI], 80% to 97%) and specificity of 97% (CI, 85% to 100%). Importantly, in HIV-negative subjects positive for TB by sputum smear microscopy, the test achieved a sensitivity of 80% (CI, 55% to 93%). This compares to an overall sensitivity of 33% (CI, 20% to 48%) of the Alere LF-LAM and a sensitivity of 13% (CI, 4% to 38%) in HIV-negative subjects in the same sample set. The capture antibody targets a unique 5-methylthio-d-xylofuranose (MTX)-dependent epitope in LAM that is specific to the Mycobacterium tuberculosis complex and shows no cross-reactivity with fast-growing mycobacteria or other bacteria. The present study provides evidence that improved assay methods and reagents lead to increased diagnostic accuracy. The results of this work have informed the development of a sensitive and specific novel LAM point-of-care assay with the aim to meet the WHO's performance target for TB diagnosis.

The only currently commercialized point-of-care assay for tuberculosis (TB) that measures lipoarabinomannan (LAM) in urine (Alere LF-LAM) has insufficient sensitivity. We evaluated the potential of 100 novel monoclonal antibody pairs targeting a variety of LAM epitopes on a sensitive electrochemiluminescence platform to improve the diagnostic accuracy. In the screening, many antibody pairs showed high reactivity to purified LAM but performed poorly at detecting urinary LAM in clinical samples, suggesting differences in antigen structure and immunoreactivity of the different LAM sources. The 12 best antibody pairs from the screening were tested in a retrospective case-control study with urine samples from 75 adults with presumptive TB. The best antibody pair reached femtomolar analytical sensitivity for LAM detection and an overall clinical sensitivity of 93% (confidence interval [CI], 80% to 97%) and specificity of 97% (CI, 85% to 100%). Importantly, in HIV-negative subjects positive for TB by sputum smear microscopy, the test achieved a sensitivity of 80% (CI, 55% to 93%). This compares to an overall sensitivity of 33% (CI, 20% to 48%) of the Alere LF-LAM and a sensitivity of 13% (CI, 4% to 38%) in HIV-negative subjects in the same sample set. The capture antibody targets a unique 5-methylthio-d-xylofuranose (MTX)-dependent epitope in LAM that is specific to the Mycobacterium tuberculosis complex and shows no cross-reactivity with fast-growing mycobacteria or other bacteria. The present study provides evidence that improved assay methods and reagents lead to increased diagnostic accuracy. The results of this work have informed the development of a sensitive and specific novel LAM point-of-care assay with the aim to meet the WHO's performance target for TB diagnosis.

BACKGROUND. Inadequate tuberculosis (TB) diagnostics are a major hurdle in the reduction of disease burden, and accurate point-of-care tests (POCTs) are urgently needed. We assessed the diagnostic accuracy of Fujifilm SILVAMP TB lipoarabinomannan (FujiLAM) POCT for TB diagnosis in HIV-negative outpatients and compared it with Alere Determine TB LAM Ag (AlereLAM) POCT and a laboratory-based ultrasensitive electrochemiluminescence LAM research assay (EclLAM). METHODS. In this multicenter diagnostic test accuracy study, we recruited HIV-negative adults with symptoms suggestive of pulmonary TB presenting to outpatient health care centers in Peru and South Africa. Urine samples were tested using FujiLAM, AlereLAM, and EclLAM, and the diagnostic accuracy was assessed against a microbiological reference standard (MRS) and a composite reference standard. RESULTS. Three hundred seventy-two HIV-negative participants were included and the prevalence of microbiologically confirmed TB was 30%. Compared with the MRS, the sensitivities of AlereLAM, FujiLAM, and EclLAM were 10.8% (95% confidence interval [CI] 6.3%-18.0%), 53.2% (95% CI 43.9%-62.1%), and 66.7% (95% CI 57.5%-74.7%), respectively. The specificities of AlereLAM, FujiLAM, and EclLAM were 92.3% (95% CI 88.5%-95.0%), 98.9% (95% CI 96.7%-99.6%), and 98.1% (95% CI 95.6%-99.2%), respectively. Positive likelihood ratios of AlereLAM, FujiLAM, and EclLAM were 1.4, 46.2, and 34.8, respectively, and positive predictive values were 37.5%, 95.2%, and 93.7%, respectively. CONCLUSION. Compared with AlereLAM, FujiLAM detected 5 times more patients with TB in HIV-negative participants, had a high positive predictive value, and has the potential to improve rapid diagnosis of TB at the point-of-care. EclLAM demonstrated that additional sensitivity gains are possible, which highlights LAM's potential as a biomarker. Additional research is required to assess FujiLAM's performance in prospective cohorts, its cost-effectiveness, and its impact in real-world clinical settings. FUNDING. Global Health Innovative Technology Fund, the UK Department for International Development, the Dutch Ministry of Foreign Affairs, the Bill and Melinda Gates Foundation, the Australian Department of Foreign Affairs and Trade, the German Federal Ministry of Education and Research through Kreditanstalt fur Wiederaufbau, and the NIH and National Institute of Allergy and Infectious Diseases., Introduction Tuberculosis (TB) is the leading single infectious cause of death worldwide, with more than 1.5 million deaths in 2018 (1). The high rate of unreported TB (estimated at 3.0 [...]

A better understanding of all immune components involved in protecting against Mycobacterium tuberculosis infection is urgently needed to inform strategies for novel immunotherapy and tuberculosis (TB) vaccine development. Although cell-mediated immunity is critical, increasing evidence supports that antibodies also have a protective role against TB. Yet knowledge of protective antigens is limited. Analyzing sera from 97 US immigrants at various stages of M. tuberculosis infection, we showed protective in vitro and in vivo efficacy of polyclonal IgG against the M. tuberculosis capsular polysaccharide arabinomannan (AM). Using recently developed glycan arrays, we established that anti-AM IgG induced in natural infection is highly heterogeneous in its binding specificity and differs in both its reactivity to oligosaccharide motifs within AM and its functions in bacillus Calmette-Guerin vaccination and/or in controlled (latent) versus uncontrolled (TB) M. tuberculosis infection. We showed that anti-AM IgG from asymptomatic but not from diseased individuals was protective and provided data suggesting a potential role of IgG2 and specific AM oligosaccharides. Filling a gap in the current knowledge of protective antigens in humans, our data support the key role of the M. tuberculosis surface glycan AM and suggest the importance of targeting specific glycan epitopes within AM in antibody-mediated immunity against TB., Introduction Active tuberculosis (TB), the clinical manifestation of uncontrolled Mycobacterium tuberculosis (Mtb) infection, is now the leading cause of death from a single pathogen worldwide. A better understanding of all [...]

A de novo asymmetric strategy for the synthesis of d‐bradyrhizose diastereomers from an achiral ketoenolester precursor is described. Key transformations used in the stereodivergent approach include two Noyori asymmetric reductions, an Achmatowicz rearrangement, diastereoselective alkene oxidations, and the first example of a palladium(0)‐catalyzed glycosylation of a vinylogous pyranone. The isomeric composition of the bicyclic reducing sugars obtained was analyzed and their behaviour was compared to the natural product, revealing key stereocentres that impact the overall distribution. [ABSTRACT FROM AUTHOR]

Carbapenemase and extended β-lactamase-producing Klebsiella pneumoniae isolates represent a major health threat, stimulating increasing interest in immunotherapeutic approaches for combating Klebsiella infections. Lipopolysaccharide O antigen polysaccharides offer viable targets for immunotherapeutic development, and several studies have described protection with O-specific antibodies in animal models of infection. O1 antigen is produced by almost half of clinical Klebsiella isolates. The O1 polysaccharide backbone structure is known, but monoclonal antibodies raised against the O1 antigen showed varying reactivity against different isolates that could not be explained by the known structure. Reinvestigation of the structure by NMR spectroscopy revealed the presence of the reported polysaccharide backbone (glycoform O1a), as well as a previously unknown O1b glycoform composed of the O1a backbone modified with a terminal pyruvate group. The activity of the responsible pyruvyltransferase (WbbZ) was confirmed by western immunoblotting and in vitro chemoenzymatic synthesis of the O1b terminus. Bioinformatic data indicate that almost all O1 isolates possess genes required to produce both glycoforms. We describe the presence of O1ab-biosynthesis genes in other bacterial species and report a functional O1 locus on a bacteriophage genome. Homologs of wbbZ are widespread in genetic loci for the assembly of unrelated glycostructures in bacteria and yeast. In K. pneumoniae, simultaneous production of both O1 glycoforms is enabled by the lack of specificity of the ABC transporter that exports the nascent glycan, and the data reported here provide mechanistic understanding of the capacity for evolution of antigenic diversity within an important class of biomolecules produced by many bacteria. [ABSTRACT FROM AUTHOR]

Abstract: Mycobacteria, including the human pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis, produce a complex cell wall structure made of carbohydrates and lipids. The major structural element of the [...]