A Mycobacterium tuberculosis fingerprint in human breath allows tuberculosis detection.

An estimated one-third of tuberculosis (TB) cases go undiagnosed or unreported. Sputum samples, widely used for TB diagnosis, are inefficient at detecting infection in children and paucibacillary patients. Indeed, developing point-of-care biomarker-based diagnostics that are not sputum-based is a major priority for the WHO. Here, in a proof-of-concept study, we tested whether pulmonary TB can be detected by analyzing patient exhaled breath condensate (EBC) samples. We find that the presence of Mycobacterium tuberculosis (Mtb)-specific lipids, lipoarabinomannan lipoglycan, and proteins in EBCs can efficiently differentiate baseline TB patients from controls. We used EBCs to track the longitudinal effects of antibiotic treatment in pediatric TB patients. In addition, Mtb lipoarabinomannan and lipids were structurally distinct in EBCs compared to ex vivo cultured bacteria, revealing specific metabolic and biochemical states of Mtb in the human lung. This provides essential information for the rational development or improvement of diagnostic antibodies, vaccines and therapeutic drugs. Our data collectively indicate that EBC analysis can potentially facilitate clinical diagnosis of TB across patient populations and monitor treatment efficacy. This affordable, rapid and non-invasive approach seems superior to sputum assays and has the potential to be implemented at point-of-care.

A Langmuir-Blodgett Study of the Interaction between Amphotericin B and Lipids of Histoplasma capsulatum.

Histoplasma capsulatum is a dimorphic, thermal, and nutritional fungus. In the environment and at an average temperature of 28 °C, it develops as a mold that is composed of infecting particles. Once in the host or in cultures at 37 °C, it undergoes a transition into the parasitic form. In the present work, we performed chemical extraction and characterization using chromatography techniques of the associated lipid composition of the external surface of the cell wall of the mycelial phase of two isolates of the H. capsulatum: one clinical and one environmental. Several differences were evidenced in the fatty acids in the phospholipid composition. Surface pressure-area isotherms and compression module curves of the Amphotericin B and lipid extract monolayers, as well as (AmB)-lipid extract mixed monolayers were recorded. Results show a high affinity of AmB towards lipid extracts. The most stable monolayers were formed by AmB + environmental with a mass ratio of 1:3 and AmB + clinical with a mass ratio of 1:2. Knowledge of the AmB aggregation processes at a molecular level and the characterization of the lipid extracts allows the possibility to understand the interaction between the AmB and the lipid fractions of H. capsulatum.

Lipid Secretion by Parasitic Cells of Coccidioides Contributes to Disseminated Disease.

Coccidioides is a soil-borne fungal pathogen and causative agent of a human respiratory disease (coccidioidomycosis) endemic to semi-desert regions of southwestern United States, Mexico, Central and South America. Aerosolized arthroconidia inhaled by the mammalian host first undergo conversion to large parasitic cells (spherules, 80-100 µm diameter) followed by endosporulation, a process by which the contents of spherules give rise to multiple endospores. The latter are released upon rupture of the maternal spherules and establish new foci of lung infection. A novel feature of spherule maturation prior to endosporulation is the secretion of a lipid-rich, membranous cell surface layer shed in vivo during growth of the parasitic cells and secretion into liquid culture medium during in vitro growth. Chemical analysis of the culture derived spherule outer wall (SOW) fraction showed that it is composed largely of phospholipids and is enriched with saturated fatty acids, including myristic, palmitic, elaidic, oleic, and stearic acid. NMR revealed the presence of monosaccharide- and disaccharide-linked acylglycerols and sphingolipids. The major sphingolipid components are sphingosine and ceramide. Primary neutrophils derived from healthy C57BL/6 and DBA/2 mice incubated with SOW lipids revealed a significant reduction in fungicidal activity against viable Coccidioides arthroconidia compared to incubation of neutrophils with arthroconidia alone. Host cell exposure to SOW lipids had no effect on neutrophil viability. Furthermore, C57BL/6 mice that were challenged subcutaneously with Coccidioides arthroconidia in the presence of the isolated SOW fraction developed disseminated disease, while control mice challenged with arthroconidia alone by the same route showed no dissemination of infection. We hypothesize that SOW lipids contribute to suppression of inflammatory response to Coccidioides infection. Studies are underway to characterize the immunosuppressive mechanism(s) of SOW lipids.

Fatty acid derivative, chemokine, and cytokine profiles in exhaled breath condensates can differentiate adult and children paucibacillary tuberculosis patients.

The anti-mycobacterial immune response in adults and children with tuberculosis (TB), as well as the response in bacteriologically positive and negative patients, is different. However, knowledge of the immunological events occurring in the lungs in these clinical situations remains scarce. Exhaled breath condensate (EBC) samples may be useful for studying the inflammatory environment of the lower airways in TB patients. The fatty acid, cytokine, and chemokine profiles in EBC from healthy adults; smear-positive and smear-negative adult patients; and healthy, asthmatic, and TB children were determined using gas chromatography and LUMINEX, respectively. Unsaturated fatty acids, particularly oleate, were increased in TB adults and children compared with healthy individuals. Elevated levels of IL-17 were characteristic of paucibacillary patients (adults and children), whereas elevated MCP-1 (monocyte chemotactic protein-1) levels were characteristic of adult patients (smear-positive and smear-negative). The levels of all of the molecules were comparable to the controls after anti-TB treatment, suggesting that changes in the levels of the molecules detected in the EBC samples were the result of the active pulmonary TB. EBC samples may be an important tool for the detection of potential early biomarkers in the different clinical manifestations of pulmonary TB and a useful tool for the diagnosis of TB, particularly in children.

Mononuclear phagocyte accumulates a stearic acid derivative during differentiation into macrophages. Effects of stearic acid on macrophage differentiation and Mycobacterium tuberculosis control.

The fatty acid composition of monocytes changes substantially during differentiation into macrophages, increasing the proportion of saturated fatty acids. These changes prompted us to investigate whether fatty acid accumulation in the extracellular milieu could affect the differentiation of bystander mononuclear phagocytes. An esterified fatty acid derivative, stearate, was the only fatty acid that significantly increased in macrophage supernatants, and there were higher levels when cells differentiated in the presence of Mycobacterium tuberculosis H37Rv or purified protein derivative (PPD). Exogenous stearic acid enhanced the expression of HLA-DR and CD64; there was also accumulation of IL-12, TNF-α, IL-6, MIP-1 α and ß and a reduction in MCP-1 and the bacterial load. These results suggested that during differentiation, a derivative of stearic acid, which promotes the process as well as the effector mechanisms of phagocytes against the mycobacterium, accumulates in the cell supernatants.

Evaluación de la actividad biológica de extractos de semillas de Crotalaria pallida (cascabelito) sobre el modelo Drosophila melanogaster / Evaluation of the biological activity of extracts from Crotalaria pallida (streaked rattlepod) seeds using the Drosophila melanogaster model

Introducción: la búsqueda de metabolitos de origen natural, con actividad biológica promisoria, particularmente la actividad insecticida, es un blanco interesante en las investigaciones sobre productos naturales. Objetivos: evaluar la bioactividad de extractos de diferente polaridad de semillas de Crotalaria pallida Aiton sobre el modelo biológico Drosophila melanogaster. Métodos: la bioactividad de los extractos de diferente polaridad de semillas secas de C. pallida se evalúo por ingestión en el modelo biológico; permitiendo purificar y determinar la estructura química del principio activo usando RMN. Resultados: la bioactividad expresada resultó de dos tipos; uno causó la inhibición de los estados larvarios, evidenciada con la disminución del número de pupas de los tratamientos con respecto a los controles no tratados, la relación dosis-respuesta permitió calcular una CI 50 de 156,47 ppm; el otro efecto inhibió el paso pupa-adulto, disminuyendo el número de adultos de los tratamientos frente a los controles, estableciéndose una CI50 de 7,95 ppm. Con el uso de diferentes ensayos de RMN se determinó el alcaloide usaramina como responsable de esta actividad biológica. Conclusiones : la bioactividad de los extractos de polaridad media/baja permitió el aislamiento de un metabolito con actividad insecticida promisoria, manifestada con la inhibición del normal desarrollo del ciclo de vida de D. melanogaster, el extracto no exhibe actividad sobre la oviposición, en el intervalo de concentraciones evaluado; a bajas concentraciones inhibe la eclosión de pupas y a altas concentraciones afecta el desarrollo de las larvas; actividades que se mantienen al probar el metabolito purificado.

Introduction: the search for metabolites of natural origin with promising biological -particularly insecticidal- activity, is an interesting target for research about natural products. Objectives: evaluate the bioactivity of extracts of varying polarity from Crotalaria pallida Aiton seeds using the Drosophila melanogaster biological model. Methods: an evaluation was conducted of the bioactivity of extracts of varying polarity from dry C. pallida seeds by ingestion into the biological model, with the purpose of purifying and determining the chemical structure of the active principle through NMR spectroscopy. Results: two types of bioactivity were expressed. One caused inhibition of larval stages, evidenced in a smaller number of pupae in treatment controls with respect to non-treatment controls. The dose-response relationship allowed estimation of a CI50 of 156.47 ppm. The other effect inhibited progress from pupa to adult, reducing the number of adults in the treatment vs. control groups, with an CI50 of 7.95 ppm. With the use of various NMR assays, it was determined that the alkaloid usaramine was responsible for this biological activity. Conclusions: the bioactivity of extracts of medium / low polarity permitted the isolation of a metabolite with promising insecticidal activity, manifested in the inhibition of the normal development of the life cycle of D. melanogaster. The extract does not show any activity on oviposition in the concentration range studied. At low concentrations it inhibits the eclosion of pupae, whereas at high concentrations it affects larval development. Both activities remained when the purified metabolite was tested.

Fungicidal activity of Eucalyptus tereticornis essential oil on the pathogenic fungus Fusarium oxysporum / Actividad antimicótica del aceite esencial a partir de Eucalyptus tereticornis sobre el hongo patógeno Fusarium oxysporum

The objective of present paper was to determine the antifungal activity of the Eucalyptus tereticornis (Myrtaceae) essential oil and two fractions on the Fusarium oxysporum mushroom, a pathogen with clinical and agricultural significance. The total citronelal (44.8 percent) and geraniol (9.78 percent) essential oil had a fungicidal effect at a 3 g/L concentration and a fungicidal activity at small concentrations. The A and B fractions composed most of p-mentane-3,8-diol (18.95 percent) and geraniol acetate (24.34 percent), respectively were more active than the total extract. The observations at microscopic level showed damages and changes in hyphae and chlamydospores, as well as a decrease in the number of conidia. The observed fungicidal activity and the morphologic damages were dependent on the concentration.

El objetivo de este trabajo fue determinar la actividad antifúngica del aceite esencial de Eucalyptus tereticornis (Myrtaceae) y 2 fracciones sobre el hongo Fusarium oxysporum, patógeno de importancia tanto clínica como agrícola. El aceite esencial total, compuesto principalmente por citronelal (44,8 por ciento), citronelol (9,78 por ciento) presentó un efecto fungicida a una concentración de 3 g/L y actividad fungistática a concentraciones menores. La fracciones A y B compuestas en su mayoría por p-mentano-3,8-diol (18,95 por ciento) y acetato de citronelol (24,34 por ciento) respectivamente fueron más activas que el extracto total. Las observaciones a nivel microscópico mostraron daños y cambios en hifas y clamidosporas, así como disminución en el número de conidias. La actividad fungistática observada y los daños morfológicos fueron dependientes de la concentración.