eNose analysis of volatile chemicals from dogs naturally infected with Leishmania infantum in Brazil.

BACKGROUND: Visceral leishmaniasis (VL) in Brazil is a neglected, vector-borne, tropical parasitic disease that is responsible for several thousand human deaths every year. The transmission route involves sand flies becoming infected after feeding on infected reservoir host, mainly dogs, and then transmitting the Leishmania infantum parasites while feeding on humans. A major component of the VL control effort is the identification and euthanasia of infected dogs to remove them as a source of infection. A rapid, non-invasive, point-of-care device able to differentiate between the odours of infected and uninfected dogs may contribute towards the accurate diagnosis of canine VL. METHODOLOGY/PRINCIPAL FINDINGS: We analysed the headspace volatile chemicals from the hair of two groups of dogs collected in 2017 and 2018 using a bench-top eNose volatile organic chemical analyser. The dogs were categorised as infected or uninfected by PCR analysis of blood samples taken by venepuncture and the number of parasites per ml of blood was calculated for each dog by qPCR analysis. We demonstrated using a robust clustering analysis that the eNose data could be discriminated into infected and uninfected categories with specificity >94% and sensitivity >97%. The eNose device and data analysis were sufficiently sensitive to be able to identify infected dogs even when the Leishmania population in the circulating blood was very low. CONCLUSIONS/SIGNIFICANCE: The study illustrates the potential of the eNose to rapidly and accurately identify dogs infected with Le. infantum. Future improvements to eNose analyser sensor sensitivity, sampling methodology and portability suggest that this approach could significantly improve the diagnosis of VL infected dogs in Brazil with additional potential for effective diagnosis of VL in humans as well as for the diagnosis of other parasitic diseases.

Chemical modification and immobilisation of laccase from Trametes hirsuta and from Myceliophthora thermophila.

Laccase from two different source organisms, Myceliophthora thermophila and Trametes hirsuta, were subjected to chemical modification in solution by (i) two bifunctional reagents, ethylene-glycol-N-hydroxy succinimide (EGNHS) and glutaraldehyde and (ii) by the monofunctional citraconic anhydride. The untreated and chemically modified forms of both enzymes were then immobilised onto three different types of mesoporous silicate (MPS) particle (MCM, CNS and SBA-15). Thermal stabilities of native, modified-soluble and immobilised laccases were then evaluated. Although the two laccases have similar lysine contents, those of M. thermophila are clearly more amenable to chemical modification. Treatment of the M. thermophila enzyme with EGNHS led to a 8.7-fold increase in thermal stability over the free soluble enzyme while glutaraldehyde gave a 5.7-fold increase. Increased activity of M. thermophila laccase occurred only with citraconic anhydride modification (a 3-fold increase), while the glutaraldehyde modification marginally increased the activity of the T. hirsuta enzyme (by 1.2-fold). Upon immobilisation onto MPS, the greatest increase in stability was for the glutaraldehyde-treated M. thermophila preparation on SBA-15 (24-fold over the soluble enzyme). Chemical modification of laccase from T. hirsuta with both glutaraldehyde and EGNHS gave only a 2-fold increase in stability, increasing >4-fold upon immobilisation onto SBA-15 and MCM-41/98.

Discrimination of plant volatile signatures by an electronic nose: aA potential technology for plant pest and disease monitoring.

The volatile organic compounds (VOCs) profile emitted from plants often changes in response to environmental factors, and monitoring the change of such profiles could provide a nondestructive means of plant health measurement An electronic nose (e-nose) was used to discriminate among VOC bouquets emitted by cucumber, pepper, and tomato leaves subjected to mechanical damage or pest and disease attacks compared with undamaged control leaves. Principle component analysis, discriminant function analysis, and cluster analysis were applied to evaluate the data. The results indicate that the e-nose can discriminate among VOCs from undamaged leaves of the three tested species. It can also discriminate undamaged and artificially damaged leaves of the same plant species. In cucumber, the e-nose can discriminate among VOCs emitted from control, artificially damaged, and spider-mite-infested leaves. It could also discriminate among VOCs emitted from control, artificially damaged, hornworm-damaged, and powdery-mildew-infected tomato leaves. The relationships between the changes in volatile signatures detected by the e-nose to changes in the underlying chemistry of plant VOC signatures in response to applied stresses were quantified by gas chromatography mass spectrometry. We conclude that the e-nose had genuine responses to changes in plant VOC signatures and can successfully discriminate them. These studies demonstrate the potential use of such e-nose technology as a real time pest and disease monitoring system in agricultural and horticultural settings.

Label-less immunosensor assay for myelin basic protein based upon an ac impedance protocol.

This paper describes the development and characterization of a label-less immunosensor for myelin basic protein (MBP) and its interrogation using an ac impedance protocol. Commercial screen-printed carbon electrodes were used as the basis for the sensor. Polyaniline was electrodeposited onto the sensors, and this modified surface then utilized to immobilize a biotinylated antibody for MBP using a classical avidin-biotin approach. Electrodes containing the antibodies were exposed to solutions of MBP and interrogated using an ac impedance protocol. The real component of the impedance of the electrodes was found to increase with increasing concentration of antigen. Control samples containing a nonspecific IgG antibody were also studied, and calibration curves were obtained by subtraction of the responses for specific and nonspecific antibody-based sensors, thereby accounting for and eliminating the effects of nonspecific adsorption of MBP. A logarithmic relationship between the concentration of MBP in buffer solutions and the impedimetric response was observed.

Detection of fluoroquinolone antibiotics in milk via a labeless immunoassay based upon an alternating current impedance protocol.

This paper describes the construction of a labeless immunosensor for the antibiotic ciprofloxacin in milk and its interrogation using an ac impedance protocol. Commercial screen-printed carbon electrodes were used as the basis for the sensor. Polyaniline was electrodeposited onto the sensors and then utilized to immobilize a biotinylated antibody for ciprofloxacin using classical avidin-biotin interactions. Antibody loaded electrodes were exposed to solutions of antigen in milk and interrogated using an ac impedance protocol. The faradaic component of the impedance of the electrodes was found to increase with increasing concentration of antigen. Control samples containing a nonspecific IgG antibody were also studied but were found to display large nonspecific responses, probably due to the antibody binding some of the large number of components found in milk. Control sensors could, however, be fabricated using antibodies specific for species not found in milk. Calibration curves could be obtained by subtraction of the responses for specific and control antibody-based sensors, thereby eliminating the effects of nonspecific adsorption of antigen. Sensors exposed to ciprofloxacin in milk gave increases in impedance whereas ciprofloxacin in phosphate buffer led to decreases, indicating the possibility of developing sensors which can both detect and differentiate between free and chelated antigen.