No abstract

Analysis of volatile organic compounds in the breath for disease detection and monitoring has gained momentum and clinical significance due to its rapid test results and non-invasiveness, especially for diabetes mellitus (DM). Studies have suggested that breath gases, including acetone, may be related to simultaneous blood glucose (BG) and blood ketone levels in adults with types 2 and 1 diabetes. Detecting altered concentrations of ketones in the breath, blood and urine may be crucial for the diagnosis and monitoring of diabetes mellitus. This study assesses the efficacy of a simple breath test as a non-invasive means of diabetes monitoring in adults with type 2 diabetes mellitus. Human breath samples were collected in Tedlar™ bags and analyzed by headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME/GC-MS). The measurements were compared with capillary BG and blood ketone levels (β-hydroxybutyrate and acetoacetate) taken at the same time on a single visit to a routine hospital clinic in 30 subjects with type 2 diabetes and 28 control volunteers. Ketone bodies of diabetic subjects showed a significant increase when compared to the control subjects; however, the ketone levels were was controlled in both diabetic and non-diabetic volunteers. Worthy of note, a statistically significant relationship was found between breath acetone and blood acetoacetate (R = 0.89) and between breath acetone and β-hydroxybutyrate (R = 0.82).

Cell wall mycolic acids (MA) from Mycobacterium tuberculosis (M.tb) are CD1b presented antigens that can be used to detect antibodies as surrogate markers of active TB, even in HIV coinfected patients. The use of the complex mixtures of natural MA is complicated by an apparent antibody cross-reactivity with cholesterol. Here firstly we report three recombinant monoclonal scFv antibody fragments in the chicken germ-line antibody repertoire, which demonstrate the possibilities for cross-reactivity: the first recognized both cholesterol and mycolic acids, the second mycolic acids but not cholesterol, and the third cholesterol but not mycolic acids. Secondly, MA structure is experimentally interrogated to try to understand the cross-reactivity. Unique synthetic mycolic acids representative of the three main functional classes show varying antigenicity against human TB patient sera, depending on the functional groups present and on their stereochemistry. Oxygenated (methoxy- and keto-) mycolic acid was found to be more antigenic than alpha-mycolic acids. Synthetic methoxy-mycolic acids were the most antigenic, one containing a trans-cyclopropane apparently being somewhat more antigenic than the natural mixture. Trans-cyclopropane-containing keto- and hydroxy-mycolic acids were also found to be the most antigenic among each of these classes. However, none of the individual synthetic mycolic acids significantly and reproducibly distinguished the pooled serum of TB positive patients from that of TB negative patients better than the natural mixture of MA. This argues against the potential to improve the specificity of serodiagnosis of TB with a defined single synthetic mycolic acid antigen from this set, although sensitivity may be facilitated by using a synthetic methoxy-mycolic acid.

The appearance of drug-resistant strains of Mycobacterium tuberculosis (Mtb) poses a great challenge to the development of novel treatment programmes to combat tuberculosis. Since innovative nanotechnologies might alleviate the limitations of current therapies, we have designed a new nanoformulation for use as an anti-TB drug delivery system. It consists of incorporating mycobacterial cell wall mycolic acids (MA) as targeting ligands into a drug-encapsulating Poly dl-lactic-co-glycolic acid polymer (PLGA), via a double emulsion solvent evaporation technique. Bone marrow-derived mouse macrophages, either uninfected or infected with different mycobacterial strains (Mycobacterium avium, Mycobacterium bovis BCG or Mtb), were exposed to encapsulated isoniazid-PLGA nanoparticles (NPs) using MA as a targeting ligand. The fate of the NPs was monitored by electron microscopy. Our study showed that i) the inclusion of MA in the nanoformulations resulted in their expression on the outer surface and a significant increase in phagocytic uptake of the NPs; ii) nanoparticle-containing phagosomes were rapidly processed into phagolysosomes, whether MA had been included or not; and iii) nanoparticle-containing phagolysosomes did not fuse with non-matured mycobacterium-containing phagosomes, but fusion events with mycobacterium-containing phagolysosomes were clearly observed.