A microfluidic electrochemical biosensor based on multiwall carbon nanotube/ferrocene for genomic DNA detection of Mycobacterium tuberculosis in clinical isolates.

Herein we present a microfluidic-multiplexed platform that integrates electrochemical sensors based on carbon nanotubes associated with ferrocene as redox marker (carbon nanotube (CNT)/ferrocene) for direct detection of pathogenic viral DNA from Hepatitis C and genomic DNA from Mycobacterium tuberculosis in clinical isolates. By operating the fluidic device under high flow (150 µl/min), the formation of a very thin depletion layer at the sensor surface (δS = 230 nm) enhances the capture rate up to one DNA strand per second. By comparison, this capture rate is only 0.02 molecule/s in a static regime without flow. This fluidic protocol allows thus enhancing the limit of detection of the electrochemical biosensor from picomolar in bulk solution to femtomolar with a large dynamic range from 0.1 fM to 1 pM. Kinetics analysis also demonstrates an enhancement of the rate constant of electron transfer (kS) of the electrochemical process from 1 s(-1) up to 6 s(-1) thanks to the geometry of the miniaturized fluidic electrochemical cell. This microfluidic device working under high flow allows selective direct detection of a Mycobacterium tuberculosis (H37Rv) rpoB allele from clinical isolate extracted DNA. We envision that a microfluidic approach under high flow associated with a multiwall CNT/ferrocene sensor could find useful applications as the point-of-care for multi-target diagnostics of biomarkers in real samples.

First Report of Botryosphaeria dothidea, Diplodia seriata, and Neofusicoccum luteum Associated with Canker and Dieback of Grapevines in Tunisia.

In 2011, common symptoms of grapevine dieback were frequently observed in 2- to 5-year-old table grape (Vitis vinifera L.) cvs. in four vineyards located in northern Tunisia. The symptoms included dead spur and cordons, shoot dieback, and sunken necrotic bark lesions, which progressed into the trunk resulting in the death of large sections of the vine. Longitudinal and transversal sections of cordons and spurs from symptomatic vines revealed brown wedge-shaped cankers of hard consistency. Twelve symptomatic samples from spur and cordons were collected, surface disinfected by dipping into 5% (v/v) sodium hypochlorite for 2 min, and small pieces from the edge of necrotic and healthy tissue were removed and plated onto potato dextrose agar (PDA) at 25°C in the dark. Based on colony and conidia morphological characteristics, isolates were divided in three species, named Diplodia seriata, Botryosphaeria dothidea, and Neofusicoccum luteum. D. seriata colonies were gray-brown with dense aerial mycelium producing brown cylindric to ellipsoid conidia rounded at both ends and averaged 22.4 × 11.7 µm (n = 50). B. dothidea colonies were initially white with abundant aerial mycelium, gradually becoming dark green olivaceous. Conidia were fusiform to fusiform elliptical with a subobtuse apex and averaged 24.8 × 4.7 µm (n = 50). N. luteum colonies were initially pale to colorless, gradually darkening with age and becoming gray to dark gray producing a yellow pigment that diffuses into the agar. Conidia were hyaline, thin-walled, aseptate, fusiform to fusiform elliptical, and averaged 19.8 × 5.5 µm (n = 50). Identity of the different taxa was confirmed by sequence analyses of the internal transcribed spacer (ITS1-5.8S-ITS2) region of the rDNA and part of the elongation factor 1-alpha (EF1-α) gene. BLAST analysis of sequences indicated that six isolates were identified as D. seriata (GenBank: AY259094, AY343353), one isolate as B. dothidea (AY236949, AY786319) and one isolate as N. luteum (AY259091, AY573217). Sequences were deposited in GenBank under accessions from KC178817 to KC178824 and from KF546829 to KF546836 for ITS region and EF1-α gene, respectively. A pathogenicity test was conducted on detached green shoots cv. Italia for the eight Botryosphaeriaceae isolates. Shoots were inoculated by placing a colonized agar plug (5 mm diameter) from the margin of a 7-day-old colony on fresh wound sites made with a sterilized scalpel. Each wound was covered with moisturized cotton and sealed with Parafilm. Control shoots were inoculated using non-colonized PDA plugs. After 6 weeks, discoloration of xylem and phloem and necrosis with average length of 38.8, 17.6, and 11.2 mm were observed from inoculated shoots with D. seriata, N. luteum, and B. dothidea, respectively, and all three fungi were re-isolated from necrotic tissue, satisfying Koch's postulates. Control shoots showed no symptoms of the disease and no fungus was re-isolated. In Tunisia, Botryosphaeria-related dieback was reported only on citrus tree caused by B. ribis (2), on Pinus spp. caused by D. pinea (4), on Quercus spp. caused by D. corticola (3), and on olive tree (Olea europea) caused by D. seriata (1). To our knowledge, this is the first report of D. seriata, B. dothidea, and N. luteum associated with grapevine dieback in Tunisia. References: (1) M. Chattaoui et al. Plant Dis. 96:905, 2012. (2) H. S. Fawcett. Calif. Citrogr. 16:208, 1931. (3) B. T. Linaldeddu et al. J. Plant Pathol. 91:234. 2009. (4) B. T. Linaldeddu et al. Phytopathol. Mediterr. 47:258, 2008.

First Report of Agrobacterium vitis as Causal Agent of Crown Gall Disease of Grapevine in Tunisia.

Since October 2011, a serious outbreak of crown gall disease was observed on 1- and 2-year-old grapevines (Vitis vinifera L.) cv. Superior Seedless in several vineyards located in the region of Regueb in the center of Tunisia. Fifty isolates of Agrobacterium were isolated on a tartrate medium from galls of affected plants. To prepare template DNA, cell suspensions were lysed in 0.25% sodium-azide (NaN3) buffer prepared in 1% Triton X-100 by heating the samples at 95°C for 10 to 15 min (1). The strains were differentiated using a multiplex PCR assay with a combination of VIRFF1/VIRFR2 and VIRD2S4F716/VIRD2S4R1036 primers (2), which detect regions of virF and virD2 genes, respectively, in A. vitis strains carrying octopine or nopaline Ti plasmids and A. vitis vitopine strains. In order to differentiate A. vitis strains from A. tumefaciens strains, PGF/PGR (4), a polygalacturonase specific primer set, was added to the mixture in multiplex PCR. The isolates segregated into three main groups. The first group carries octopine type Ti plasmids, the second carries vitopine type Ti plasmids, and the third group carries both octopine and vitopine type Ti plasmids. The polygalacturonase gene sequence from 10 isolates showed 94 to 97% identity to the sequences of A. vitis previously deposited in the NCBI GenBank database (Accession No. CP000633.1gb). The biochemical test results corresponded to the results of genetic analysis. The ability to aerobically convert lactose to 3-ketolactose was tested by spotting bacteria onto medium containing lactose and flooding plates with a layer of Benedict's reagents after incubation at 28°C for 48 h. Acid production from glucose was tested by spotting bacterial strains onto potato dextrose agar (PDA) medium supplemented with CaCO3. Alkali production from L-tartrate was tested by streaking bacteria on AB minimal medium supplemented with L-tartrate and growth in salt medium was tested by streaking on nutrient broth supplemented with 2% NaCl. All isolates except one were negative in 3-ketolactose. They were negative in acid clearing on PDA-CaCO3, grew in 2% NaCl, and produced alkali from tartarate. Pathogenicity of all 50 strains was tested on 1-month-old tomato plants (Lycopersicum esculentum cv. Riograndi). Plants were inoculated on the stem by pricking one to three times through a drop of inoculum (108 CFU/ml) at three inoculation sites. Sterile distilled water was used as control treatment. Plants were grown for 4 weeks at 23 ± 3°C and symptoms were recorded. Typical tumors developed at the inoculation sites and no symptoms were observed on the control plants. In Tunisia, crown gall disease was observed only on stone fruit trees and only A. tumefaciens Biovar 1 have been reported and assigned to four genomic species G4, G6 G7, and G8 basically on the recA sequencing (3). To our knowledge, this is the first report of A. vitis determined as the causal agent of grapevine crown gall in Tunisia. References: (1) A. Abolmaaty et al. Microbios 101:181, 2000. (2) F. Bini et al. Vitis 47:181, 2008. (3) D. Costechareyre et al. Microb. Ecol. 60:862, 2010. (4) E. Szegedi and S. Bottka. Vitis 41:37, 2002.

Occurrence of ochratoxin A in domestic beers and wines from Tunisia by immunoaffinity clean-up and liquid chromatography.

A survey on the occurrence of ochratoxin A (OTA) in wines and beers produced in Tunisia was carried out. Wines and beers were analysed using immunoaffinity column clean-up and high-performance liquid chromatography coupled to a fluorometric detector. OTA was detected in 29 wine samples, with an incidence of contamination of 85%. The OTA levels ranged between 0.09 and 1.5 µg/L. Neither of the studied samples showed levels above the European regulatory limit (2 µg/L). OTA was detected in 17 beer samples with an incidence of contamination of 45%. The OTA levels ranged between 0.04 and 0.35 µg/L. The OTA dietary intake by the consumption of wine and beer may be considered as negligible. The obtained results showed high incidence of OTA in Tunisian wines and beers; however, there are no toxicological risks for Tunisian consumers through their consumption of such processed products using cereals and grapes.

Electrochemical detection of D-dimer as deep vein thrombosis marker using single-chain D-dimer antibody immobilized on functionalized polypyrrole.

We describe a rapid and sensitive method for detection and quantification of d-dimer which is a biomarker present at elevated concentrations in patients with deep vein thrombosis (DVT) disorders. The method uses an immunosensor based on a single-chain antibody (ScAb) immobilized on a transducer surface and with a densely packed receptor layer. Detection is based on the redox activity of a N-alpha bis(carboxymethyl)-L-lysine (ANTA)/Cu2+ complex attached to a polypyrrole backbone. The resulting hybrid material: polypyrrole ANTA/metal complex/His-tag ScAb was characterized by AFM, surface plasmon resonance (SPR) and differential pulse voltammetry (DPV) for the optimization of the biosensor formation. The biosensor offers a promising template for antibody immobilization and for immunodetection of a specific D-dimer. The biosensor shows a remarkable variation in redox activity of the ANTA/Cu2+ complex after the D-dimer association with a binding constant Kd of 1 ng mL(-1). Electrochemical impedance spectroscopy (EIS) allows monitoring D-dimer association with a linear response between 0.1 ng mL(-1) and 500 ng mL(-1) and a detection limit of 100 pg mL(-1) in PBS is obtained. The biolayer exhibits the same sensitivity for the detection of d-dimer in human patient plasma samples. This assay method is versatile, offers enhanced performance for the evaluation of proteins association and could easily be extended to the detection of other proteins, present in serum human sample.

Effect of the size of electrode on electrochemical properties of ferrocene-functionalized polypyrrole towards DNA sensing.

A simple and highly sensitive electrochemical DNA sensor based on a ferrocene-functionalized polypyrrole has been prepared on a microelectrode array substrate for a multi-DNA detection chip format. A copolymer formed with 1-(phthalimidylbutanoate)-1'-(N-(3-butylpyrrole)butanamide)ferrocene (Py-Fe-NHP) and pyrrole was electrocopolymerized on the gold surface of both macroelectrode and biochip formats. DNA probes bearing an amino group were covalently grafted by substitution of NHP groups and the hybridization reaction was followed by monitoring the redox signal of the ferrocenyl group acting as the probe. The integration of the polymers into chip format produces high-density arrays of individually addressable oligonucleotide microelectrodes. Results show that reducing the size of the electrodes from a macroelectrode to the chip format allows a variation of the nucleation and the growth process during electropolymerization of modified pyrrole monomers. These modifications enable an increase in the sensitivity and selectivity of DNA hybridization.

Occurrence of pathogenic fungal species in Tunisian vineyards.

Mycotoxins are secondary metabolites produced by filamentous fungi detected in food, such are grapes. OTA was evaluated in ten handle musts from different Tunisian vineyard. This mycotoxin was found at levels 1.1 mug/L to 4.3 mug/L. A survey was conducted to assess the contamination of the Tunisian vineyard with pathogenic fungal species, in particular those responsible of the OTA production. The results were evaluated for the first time in parcels cultivated in the North, in the Centre and in the South of the country. Italia Muscate and Superior Seedless varieties were concerned at three developmental stages of the berry, setting, veraison and maturity. Carigon variety was used as positive control for musts contaminating by OTA. The main fungal species isolated were Aspergillus spp. (33.32%), Botrytis cinerea (23.32%), Alternaria spp. (12.80%), Cladosporium spp. (10.59%) and Penicillium spp. (8.3%). The isolates of the Aspergillus genus were identified as Aspergillus niger aggregate (77%), Aspergillus carbonarius (15%) and Aspergillus flavus (8%). Their presence was characterized by a significant decrease in the Centre during the veraison and a slight increase in the North and the South during the maturity stage. Furthermore, when comparing Superior Seedless and Italia Muscate cultivated in the same area, the aspergilli were particularly less abundant at the setting stage in the case of Superior Seedless. There is no correlation between the OTA amount in musts and the contamination by Aspergillus species in different vineyards and for grape varieties studied.

[Infantile visceral leishmaniasis: difficult diagnosis in cases complicated by hemophagocytosis]. / Leishmaniose viscérale infantile: un diagnostic difficile en cas d'hémophagocytose associée.

BACKGROUND: Hemophagocytosis has already been reported in cases of visceral leishmaniasis and thus may complicate search for diagnosis. CASE REPORT: A previously healthy 2 year-old boy was referred for fever and splenomegaly with pancytopenia. An initial diagnosis of kala-azar was refuted because of absence of biological inflammatory syndrome, negativity of bone-marrow aspiration and splenic ponction and of specific serology. After three months of clinical deterioration and apparition of active hemophagocytosis, both bone marrow aspiration and specific serology for visceral leishmaniasis became positive. The boy was given sodium stibogluconate for 20 days; he improved gradually with complete and definitive remission. CONCLUSION: Diagnosis of visceral leishmaniasis may be difficult, even in countries where this condition is relatively frequent; the association with hemophagocytosis is possible and does not constitute a poor factor of prognosis if specific therapy is proposed.