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The diagnosis of non-tuberculous mycobacteria (NTM) is a particular challenge in people with cystic fibrosis. Current standard diagnostic approaches rely on serial sputum culture, which is resource demanding, dependent on patient expectoration and may be compromised by excessive decontamination, conventional bacterial overgrowth and masking by concomitant oral and nebulised antibiotics. An alternative rapid, reliable and inexpensive diagnostic method is therefore urgently needed. Serum of patients withMycobacterium abscessusinfection and chronic suppurative lung disease without NTM infection was tested against an array of novel synthetic mycolic acids, identical or similar to natural components of mycobacterial cell walls, and glycopeptidolipid (GPL)-core antigen, which has previously been investigated inMycobacterium aviumpulmonary infection. Diagnostic accuracy of individual antigens and combination of various antigens were calculated. An ELISA using individual trehalose dimycolates and GPL-core antigen was able to effectively distinguish serum from infected and non-infected individuals with a specificity of 88% and a sensitivity of up to 88%, which increased to 88% sensitivity and 93% specificity by combining several antigens in the test. These results suggest synthetic mycolic acid antigens, used individually or in combination with GPL-core antigen could be successfully used to distinguish patients withM. abscessusinfection from disease controls.

With nontuberculous mycobacterial pulmonary disease (NTM-PD) increasing in prevalence worldwide, there is a pressing clinical need for improved diagnostic techniques. There are several problems with the current diagnostic framework, requiring positive microbiology, supportive clinical features and characteristic radiological findings. Serology has previously been investigated as an adjunct to diagnosis of NTM-PD in several pilot studies suggesting potential benefit. We performed a case-control study investigating the diagnostic accuracy of a novel ELISA assay using synthetic antigens based on single mycolic acids, which are ubiquitous cell wall components of mycobacteria, in the diagnosis of NTM-PD. Adult patients with either cystic fibrosis or bronchiectasis were recruited, and demographic data including details of NTM-PD and previous treatment were recorded. Serum samples were taken on a single occasion from each subject. Each sample was tested against an array of antigens. Overall, 62 patients with cystic fibrosis and 29 patients with bronchiectasis of other causes were recruited, including 25 patients with NTM-PD due to M. abscessus, 17 with NTM-PD due to Mycobacterium avium complex (MAC), 9 patients with possible NTM-PD, and 40 disease controls with either cystic fibrosis or bronchiectasis. ELISA testing was variably positive to 25 individual antigens, with a sensitivity up to 88% for the M. abscessus patients and 68% for the MAC group. Specificity ranged from 77.5-90%. The results of this study suggest that serology shows promise as an adjunct to current diagnostic methods in NTM-PD. Further studies in a larger, prospective cohort are needed.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is characterized by the abundance of species specific, antigenic cell wall lipids called mycolic acids. These wax-like molecules all share an identical, amphiphilic mycolic motif, but have different functional groups in a long hydrophobic hydrocarbon mero-chain that divide them into three main classes: alpha-, keto- and methoxy-mycolic acids. Whereas alpha-mycolic acids constitutively maintain an abundance of around 50%, the ratio of methoxy- to keto-mycolic acid types may vary depending on, among other things, the growth stage of M. tuberculosis. In human patients, antibodies to mycolic acids have shown potential as diagnostic serum biomarkers for active TB. Variations in mycolic acid composition affect the antigenic properties and can potentially compromise the precision of detection of anti-mycolic acids antibodies in patient sera to natural mixtures. We demonstrate this here with combinations of synthetic mycolic acid antigens, tested against TB patient and control sera. Combinations of methoxy- and α-mycolic acids are more antigenic than combinations of keto- and α-mycolic acids, showing the former to give a more sensitive test for TB biomarker antibodies. Natural mixtures of mycolic acids isolated from mature cultures of M. tuberculosis H37Rv give the same sensitivity as that with synthetic methoxy- and α-mycolic acids in combination, in a surface plasmon resonance inhibition biosensor test. To ensure that the antigenic activity of isolates of natural mycolic acids is reproducible, we cultured M. tuberculosis H37Rv on Middlebrook 7H10 solid agar plates to stationary growth phase in a standardized, optimal way. The proportions of mycolic acid classes in various batches of the isolates prepared from these cultures were compared to a commercially available natural mycolic acid isolate. LC-MS/MS and NMR data for quantitation of mycolic acids class compositions show that the variation in batches is small, suggesting that the quality of the results for anti-mycolic acid antibody detection in the TB patients should not be affected by different batches of natural mycolic acid antigens if prepared in a standard way.

This review surveys both data obtained by the authors and published data on the partial or full hydrodehalogenation of di- and polyhalocyclopropanes (chlorides and bromides) with Grignard reagents catalyzed by titanium or zirconium compounds. The factors affecting the efficiency and selectivity of the hydrodebromination of bromocyclopropanes are considered: the nature of Grignard reagents (including isotopically labeled reagents), their transformations and effects in catalyzed and uncatalyzed reactions, the participation of solvents, catalytic and stoichiometric amounts of the catalyst, etc. A scheme is proposed in which the key steps of the mechanism of hydrodebromination of bromocyclopropanes includes three blocks of reactions: (a) the generation of a catalytically active Ti(II) species; (b) the hydrodehalogenation of bromocyclopropanes involving electron transfer from a low-valent catalyst species, formation of the cyclopropyl radical, and stabilization of this radical as a result of hydrogen atom transfer from the solvent molecule; and (c) transformations of previously formed radical species, such as dimerization and disproportionation (for example, of radical species generated from Grignard reagents or ether molecules) or the linking of alkyl radicals to radical species produced from solvent molecules.

This review surveys both data obtained by the authors and published data on the partial or full hydrodehalogenation of di- and polyhalocyclopropanes (chlorides and bromides) with Grignard reagents catalyzed by titanium or zirconium compounds. The factors affecting the efficiency and selectivity of the hydrodebromination of bromocyclopropanes are considered: the nature of Grignard reagents (including isotopically labeled reagents), their transformations and effects in catalyzed and uncatalyzed reactions, the participation of solvents, catalytic and stoichiometric amounts of the catalyst, etc. A scheme is proposed in which the key steps of the mechanism of hydrodebromination of bromocyclopropanes includes three blocks of reactions: (a) the generation of a catalytically active Ti(II) species; (b) the hydrodehalogenation of bromocyclopropanes involving electron transfer from a low-valent catalyst species, formation of the cyclopropyl radical, and stabilization of this radical as a result of hydrogen atom transfer from the solvent molecule; and (c) transformations of previously formed radical species, such as dimerization and disproportionation (for example, of radical species generated from Grignard reagents or ether molecules) or the linking of alkyl radicals to radical species produced from solvent molecules.