Nile red staining for rapid screening of plastic-suspect particles in edible seafood tissues.

Concerns regarding microplastic (MP) contamination in aquatic ecosystems and its impact on seafood require a better understanding of human dietary MP exposure including extensive monitoring. While conventional techniques for MP analysis like infrared or Raman microspectroscopy provide detailed particle information, they are limited by low sample throughput, particularly when dealing with high particle numbers in seafood due to matrix-related residues. Consequently, more rapid techniques need to be developed to meet the requirements of large-scale monitoring. This study focused on semi-automated fluorescence imaging analysis after Nile red staining for rapid MP screening in seafood. By implementing RGB-based fluorescence threshold values, the need for high operator expertise to prevent misclassification was addressed. Food-relevant MP was identified with over 95% probability and differentiated from natural polymers with a 1% error rate. Comparison with laser direct infrared imaging (LDIR), a state-of-the-art method for rapid MP analysis, showed similar particle counts, indicating plausible results. However, highly variable recovery rates attributed to inhomogeneous particle spiking experiments highlight the need for future development of certified reference material including sample preparation. The proposed method demonstrated suitability of high throughput analysis for seafood samples, requiring 0.02-0.06 h/cm2 filter surface compared to 4.5-14.7 h/cm with LDIR analysis. Overall, the method holds promise as a screening tool for more accurate yet resource-intensive MP analysis methods such as spectroscopic or thermoanalytical techniques.

Fate and occurrence of indoxacarb during radish cultivation for multi-risk assessment.

Agrochemical indoxacarb is an important tool for selective pest control in radish that be consumed globally. A rapid and sensitive analytical method UHPLC-MS/MS was developed for tracing indoxacarb in radish leaves and roots with LOQ of 0.001 mg/kg and RT within 2 min, which were confirmed the satisfied storage stability of indoxacarb in radish matrixes with degradation rates less than 30 %. The occurrence, pharmacokinetics dissipation and concentration variation of indoxacarb were reflected by the original deposition of 2.23-4.12 mg/kg, half-lives of 2.6-8.0 d and terminal magnitude of 0.17 × 10-2-25.46 mg/kg in radish, and the influencing factors were further illustrated in terms of climate factors, crop cultivars and soil properties. The highest residues of indoxacarb were 25.46 mg/kg in leaves and 0.12 mg/kg in roots, which were higher than international maximum residue limits. A probabilistic model, as well as deterministic model, were introduced to evaluated the health risks of indoxacarb offering a better description for uncertainty. The total chronic dietary risk values of indoxacarb were 146.961-482.065 % in 12 registered crops, of which ADI % in radish was accounted for 19.8 % with risk dilution effects. The unacceptable acute dietary risks of 121.358-220.331 % were observed at 99.9th percentile, whereas the high-potential non-carcinogenic effects were observed over 90th percentile (105.035-1121.943 %). The health risks should be continuously emphasized given the increasing applications and persistent characteristics of indoxacarb, which is vital to protect the human population from hazardous effects, particularly for vulnerable children.

Bees prefer foods containing neonicotinoid pesticides.

The impact of neonicotinoid insecticides on insect pollinators is highly controversial. Sublethal concentrations alter the behaviour of social bees and reduce survival of entire colonies. However, critics argue that the reported negative effects only arise from neonicotinoid concentrations that are greater than those found in the nectar and pollen of pesticide-treated plants. Furthermore, it has been suggested that bees could choose to forage on other available flowers and hence avoid or dilute exposure. Here, using a two-choice feeding assay, we show that the honeybee, Apis mellifera, and the buff-tailed bumblebee, Bombus terrestris, do not avoid nectar-relevant concentrations of three of the most commonly used neonicotinoids, imidacloprid (IMD), thiamethoxam (TMX), and clothianidin (CLO), in food. Moreover, bees of both species prefer to eat more of sucrose solutions laced with IMD or TMX than sucrose alone. Stimulation with IMD, TMX and CLO neither elicited spiking responses from gustatory neurons in the bees' mouthparts, nor inhibited the responses of sucrose-sensitive neurons. Our data indicate that bees cannot taste neonicotinoids and are not repelled by them. Instead, bees preferred solutions containing IMD or TMX, even though the consumption of these pesticides caused them to eat less food overall. This work shows that bees cannot control their exposure to neonicotinoids in food and implies that treating flowering crops with IMD and TMX presents a sizeable hazard to foraging bees.

Box-Behnken design aided optimization and validation of developed reverse phase HPLC analytical method for simultaneous quantification of dolutegravir sodium and lamivudine co-loaded in nano-liposomes.

A stability-indicating reversed-phase high-performance liquid chromatography method for simultaneous estimation of dolutegravir sodium and lamivudine encapsulated in the nanoliposomal formulation was developed. The chromatographic parameters namely, organic phase ratio, flow rate, and sample injection volume were selected as independent factors and were optimized by multivariate Box-Behnken design. Responses analyzed were retention time, peak area, and resolution. The optimized chromatographic method with Hypersil BDS C8 CN column as stationary phase and methanol and acetonitrile mixture and acidified Milli-Q water (pH 2.8, adjusted with 0.02% v/v orthophosphoric acid) as the mobile phase in an isocratic elution mode was validated according to parameters of International Conference on Harmonization Q1(R2) guidelines. The validated reversed-phase high-performance liquid chromatography method exhibited specificity for both dolutegravir sodium and lamivudine in the presence of degradation products as well as the liposomal matrix. This method was effectively utilized to determine the amount of drug entrapped and drug loading efficiency of dolutegravir sodium and lamivudine in a nano-liposomal formulation.

Ratiometric Strategy for Electrochemical Sensing of Carbaryl Residue in Water and Vegetable Samples.

Accurate analysis of pesticide residue in real samples is essential for food safety and environmental protection. However, a traditional electrochemical sensor based on single-signal output is easily affected by background noise, environmental conditions, electrode diversity, and a complex matrix of samples, leading to extremely low accuracy. Hence, in this paper, a ratiometric strategy based on dual-signal output was adopted to build inner correction for sensing of widely-used carbaryl (CBL) for the first time. By comparison, Nile blue A (NB) was selected as reference probe, due to its well-defined peak, few effects on the target peak of CBL, and excellent stability. The effects of a derivatization method, technique mode, and pH were also investigated. Then the performance of the proposed ratiometric sensor was assessed in terms of three aspects including the elimination of system noise, electrode deviation and matrix effect. Compared with traditional single-signal sensor, the ratiometric sensor showed a much better linear correlation coefficient (r > 0.99), reproducibility (RSD < 10%), and limit of detection (LOD = 1.0 µM). The results indicated the introduction of proper reference probe could ensure the interdependence of target and reference signal on the same sensing environment, thus inner correction was fulfilled, which provided a promising tool for accurate analysis.

Residue analysis of tebufenozide and indoxacarb in chicken muscle, milk, egg and aquatic animal products using liquid chromatography-tandem mass spectrometry.

We developed an analytical method using liquid-liquid extraction (LLE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and quantify tebufenozide (TEB) and indoxacarb (IND) residues in animal and aquatic products (chicken muscle, milk, egg, eel, flatfish, and shrimp). The target compounds were extracted using 1% acetic acid (0.1% acetic acid for egg only) in acetonitrile and purified using n-hexane. The analytes were separated on a Gemini-NX C18 column using (a) distilled water with 0.1% formic acid and 5 mm ammonium acetate and (b) methanol with 0.1% formic acid as the mobile phase. All six-point matrix-matched calibration curves showed good linearity with coefficients of determination (R2 ) ≥0.9864 over a concentration range of 5-50 µg/kg. Intra- and inter-day accuracy was expressed as the recovery rate at three spiking levels and ranged between 73.22 and 114.93% in all matrices, with a relative standard deviation (RSD, corresponding to precision) ≤13.87%. The limits of quantification (LOQ) of all target analytes ranged from 2 to 20 µg/kg, which were substantially lower than the maximum residue limits (MRLs) specified by the regulatory agencies of different countries. All samples were collected from different markets in Seoul, Republic of Korea, and tested negative for tebufenozide and indoxacarb residues. These results show that the method developed is robust and may be a promising tool to detect trace levels of the target analytes in animal products.

Dissipation of neonicotinoid insecticides imidacloprid, indoxacarb and thiamethoxam on pomegranate (Punica granatum L.).

Neonicotinoid insecticides such as imidacloprid, indoxacarb and thiamethoxam are widely used for control of a large number of insect pests of pomegranate crop. Their residue levels were evaluated on pomegranate fruits over 2 years during the same cropping season. The QuEChERS analytical method in conjunction with LC-MS/MS was validated to analyse the insecticides on pomegranate fruits with peel (whole fruit), without peel (aril) and in the field soil. The method performance was satisfactory with the limit of quantification (LOQ) of 0.005 mg/kg which was below the maximum residue limits (MRLs) in pomegranate for the 3 compounds. A first order reaction kinetics was observed for the three insecticides with the half -life of degradation of 8-11.1 days for imidacloprid; 7.4-8.4 days for indoxacarb and 9.8-14.2 days for thiamethoxam. Though the insecticides are systemic in nature, the residues in the edible pomegranate aril were always < LOQ. The maximum residue levels of imidacloprid on pomegranate was less than its MRL of 1 mg/kg, so the pre-harvest interval (PHI) required was 1 day only. For indoxacarb, 31-42 days PHI was needed for the residues to reduce to its MRL of 0.02 mg/kg. The PHI of thiamethoxam was 46-77 days, the time required for its residues to reduce to its MRL of 0.01 mg/kg. Higher rainfall possibly facilitated faster dissipation of imidacloprid residues from pomegranate whereas indoxacarb and thiamethoxam remained unaffected. The results of the study can be utilized to incorporate these three chemicals in the plant protection program of pomegranate and fixation of MRL in India.

Small Molecule Interferences in Resazurin and MTT-Based Metabolic Assays in the Absence of Cells.

In vitro assays (such as resazurin and MTT) provide an opportunity to determine the cytotoxicity of novel therapeutics before moving forward with expensive and resource-intensive in vivo studies. A concern with using these assays, however, is the production of false responses in the presence of particular chemical functionalities. To better understand this phenomenon, 19 small molecules at 6 concentrations (1 µM-100 mM) were tested in the presence of resazurin and MTT reagents to highlight potential interfering species. Through the use of absorbance measurements (using well-plate assays and UV-vis spectroscopy) with parallel MS analysis, we have shown that significant conversion of the assay reagents readily occurs in the presence of many tested interfering species without the need for any cellular activity. The most attributable sources of interference seem to arise from the presence of thiol and carboxylic acid moieties. Interestingly, the detectable interferences were more prevalent and larger in the presence of MTT (19 species with some deviations >3000%) compared to resazurin (16 species with largest deviation of ∼150%). Additionally, those deviations in the presence of resazurin were only substantial at high concentrations, while MTT showed deviations across the tested concentrations. This comprehensive study gives insight into chemical functional groups (thiols, amines, amides, carboxylic acids) that may interfere with resazurin and MTT assays in the absence of metabolic activity and indicates that proper control studies must be performed to obtain accurate data from these in vitro assays.

Metabolic reduction of resazurin; location within the cell for cytotoxicity assays.

Resazurin is widely used as a metabolic indicator for living cells, however, there has been considerable debate in the literature with regards to the specific location in the cell where the non-fluorescent resazurin is reduced to the strongly fluorescent resorufin. This lack of clarity about the reduction site makes the use of resazurin reduction data in cytotoxicity studies difficult to interpret. In this study, E. faecalis, a Gram-positive and facultative anaerobic bacterial strain, and the most toxic chlorophenol, pentachlorophenol (PCP), were chosen as models for an anaerobe and toxicant, respectively. By studying the kinetics of resazurin reduction by E. faecalis after different treatments (cell disruption, bacterial filtration, and pre-exposure to toxicant), we confirmed that resazurin reduction to resorufin by live Gram-positive and facultative anaerobic bacterial cells can only happen intracellularly under anaerobic conditions, while resorufin reduction to dihydroresorufin can happen both intracellularly and extracellularly. Based on the understanding of these fundamental mechanisms, we suggest that resazurin reduction can be used as a quick bioassay for measuring cytotoxicity.

Degradation and residues of indoxacarb enantiomers in rice plants, rice hulls and brown rice using enriched S-indoxacarb formulation and enantiopure formulation.

A chiral liquid chromatography-tandem high resolution mass spectroscopic method was developed for the analysis of indoxacarb enantiomers in rice plants, rice hulls and brown rice. Chiral separation of two enantiomers was carried out on a Superchiral S-OD column maintained at 20°C and eluted with 0.3 mL/min methanol. Samples were extracted by acetonitrile solution with ultrasound and cleansed by dispersive solid-phase extraction of 50 mg of primary secondary amine and 50 mg of C18 . This method was successfully used to study the degradation and residues of two enantiomers with enriched S-indoxacarb (2.33S/1R) and pure S-indoxacarb in rice plants. The half-lives of R-indoxacarb and S-indoxacarb were 4.20-4.33 and 3.45-3.57 days in rice plants during the degradation of enriched S-indoxacarb in Guizhou and Hunan, respectively, whereas the half-lives of pure S-indoxacarb were 2.68 and 3.69 days in Guizhou and Hunan, respectively. The results indicated that preferential S-indoxacarb degradation occurred and that enantiomeric transformation was absent in the total experiment periods of pure S-indoxacarb in rice plants. The final residue concentrations of indoxacarb enantiomers in brown rice were significantly less than those in rice plants and rice hulls in the same rice field after applying indoxacarb SC and indoxacarb EC.

Simultaneous determination of residues of thiamethoxam and its metabolite clothianidin in tobacco leaf and soil using liquid chromatography-tandem mass spectrometry.

A simple analytical method was developed to simultaneously determine thiamethoxam and its metabolite, clothianidin, in fresh tobacco leaf, soil and cured tobacco leaf using liquid chromatography with tandem mass spectrometry. Thiamethoxam and clothianidin in tobacco and soil samples were extracted with acetonitrile containing 0.1% formic acid and purified using an NH2 -SPE column. The optimized method provided good linearity with coefficients of determination R2 ≥ 0.9981. The limits of detection and quantification were between 0.006-0.12 and 0.02-0.4 mg/kg, respectively. Intra- and inter-day recovery assays were used to validate the established method. The average recoveries of thiamethoxam and clothianidin in fresh tobacco leaf, soil and cured tobacco leaf were 75.04-100.47%, 75.86-86.40% and 89.83-99.39%, respectively. The intra- and inter-day relative standard deviations were all <9%. The developed method was successfully applied for the analysis of thiamethoxam and clothianidin residues in actual tobacco and soil samples. The results indicated that the established method met the requirements for the analysis of trace amounts of thiamethoxam and clothianidin in fresh tobacco leaf, soil and cured tobacco leaf.

Leaching and sorption of neonicotinoid insecticides and fungicides from seed coatings.

Seed coatings are a treatment used on a variety of crops to improve production and offer protection against pests and fungal outbreaks. The leaching of the active ingredients associated with the seed coatings and the sorption to soil was evaluated under laboratory conditions using commercially available corn and soybean seeds to study the fate and transport of these pesticides under controlled conditions. The active ingredients (AI) included one neonicotinoid insecticide (thiamethoxam) and five fungicides (azoxystrobin, fludioxonil, metalaxyl, sedaxane thiabendazole). An aqueous leaching experiment was conducted with treated corn and soybean seeds. Leaching potential was a function of solubility and seed type. The leaching of fludioxonil, was dependent on seed type with a shorter time to equilibrium on the corn compared to the soybean seeds. Sorption experiments with the treated seeds and a solution of the AIs were conducted using three different soil types. Sorption behavior was a function of soil organic matter as well as seed type. For most AIs, a negative relationship was observed between the aqueous concentration and the log Koc. Sorption to all soils tested was limited for the hydrophilic pesticides thiamethoxam and metalaxyl. However, partitioning for the more hydrophobic fungicides was dependent on both seed type and soil properties. The mobility of fludioxonil in the sorption experiment varied by seed type indicating that the adjuvants associated with the seed coating could potentially play a role in the environmental fate of fludioxonil. This is the first study to assess, under laboratory conditions, the fate of pesticides associated with seed coatings using commercially available treated seeds. This information can be used to understand how alterations in agricultural practices (e.g., increasing use of seed treatments) can impact the exposure (concentration and duration) and potential effects of these chemicals to aquatic and terrestrial organisms.

Determination of thiamethoxam residues in banana stem and fruit through LC-MS/MS.

An analytical method based on liquid chromatography coupled with mass spectroscopy/mass spectroscopy was developed and validated for the determination of thiamethoxam residues in banana fruit and stem tissue samples. In this study, Waters Alliance LC and Acquity TQD were used with an electrospray ionization interface in the positive ion mode. An isocratic flow of 0.5% HCOOH in water and 0.05% HCOOH in CH3CN was used for separation. Thiamethoxam residue was extracted from the samples using CH3CN and a dispersive solid-phase extraction method was used for subsequent cleanup. Linearity studies were conducted between 0.001 and 0.1 µg mL-1 of standard solution with three replicates for each concentration. Satisfactory recoveries (107.21 to 115.16% and 90.94 to 109.22%) and high precision (relative standard deviations of 3.71 to 12.83% and 3.24 to 10.78%) were obtained for the banana stem and banana fruit matrix, respectively. The lower limits of detection and quantification achieved were 0.002 and 0.008 µg g-1 for banana stem and 0.001and 0.005 µg g-1for banana fruit, respectively. The developed method was used to analyze the banana stem and fruit samples collected from thiamethoxam-treated fields and stems from the local market.

Spectrally Resolved, Functional Super-Resolution Microscopy Reveals Nanoscale Compositional Heterogeneity in Live-Cell Membranes.

By recording the full fluorescence spectra and super-resolved positions of ∼106 individual polarity-sensing solvatochromic molecules, we reveal compositional heterogeneity in the membranes of live mammalian cells with single-molecule sensitivity and ∼30 nm spatial resolution. This allowed us to unveil distinct polarity characteristics of the plasma membrane and the membranes of nanoscale intracellular organelles, a result we found to be due to differences in cholesterol levels. Within the plasma membrane, we observed the formation of low-polarity, raft-like nanodomains upon cholesterol addition or cholera-toxin treatment, but found this nanoscale phase separation absent in native cells. The ultimate sensitivity achieved through examining the spectra of individual molecules thus opens the door to functional interrogations of intracellular physicochemical parameters at the nanoscale.

Antifungal Susceptibility Testing of Malassezia spp. with an Optimized Colorimetric Broth Microdilution Method.

Malassezia is a genus of lipid-dependent yeasts. It is associated with common skin diseases such as pityriasis versicolor and atopic dermatitis and can cause systemic infections in immunocompromised individuals. Owing to the slow growth and lipid requirements of these fastidious yeasts, convenient and reliable antifungal drug susceptibility testing assays for Malassezia spp. are not widely available. Therefore, we optimized a broth microdilution assay for the testing of Malassezia that is based on the CLSI and EUCAST assays for Candida and other yeasts. The addition of ingredients such as lipids and esculin provided a broth medium formulation that enabled the growth of all Malassezia spp. and could be read, with the colorimetric indicator resazurin, by visual and fluorescence readings. We tested the susceptibility of 52 strains of 13 Malassezia species to 11 commonly used antifungals. MIC values determined by visual readings were in good agreement with MIC values determined by fluorescence readings. The lowest MICs were found for the azoles itraconazole, posaconazole, and voriconazole, with MIC90 values of 0.03 to 1.0 µg/ml, 0.06 to 0.5 µg/ml, and 0.03 to 2.0 µg/ml, respectively. All Malassezia spp. were resistant to echinocandins and griseofulvin. Some Malassezia spp. also showed high MIC values for ketoconazole, which is the most widely recommended topical antifungal to treat Malassezia skin infections. In summary, our assay enables the fast and reliable susceptibility testing of Malassezia spp. with a large panel of different antifungals.

Combined orcein and martius scarlet blue (OMSB) staining for qualitative and quantitative analyses of atherosclerotic plaques in brachiocephalic arteries in apoE/LDLR-/- mice.

Numerous cellular and extracellular components should be analyzed in sections of atherosclerotic plaques to assess atherosclerosis progression and vulnerability. Here, we combined orcein (O) staining for elastic fibers and martius scarlet blue (MSB) polychrome to visualize various morphological contents of plaque in brachiocephalic arteries (BCA) of apoE/LDLR-/- mice. Elastic fibers (including broken elastic laminae and 'buried' fibrous caps) were stained purple and they could be easily distinguished from collagen fibers (blue). Orcein allowed clear identification of even the finest elastic fibers. Erythrocytes were stained yellow and they could easily be discerned from mature fibrin (red). Old fibrin tends to acquire blue color. The method of OMSB staining is simple, takes less than 1 h to perform and can be adapted to automatic stainers. Most importantly, the color separation is good enough to allow digital automatic segmentation of specific components in tissue section and quantitative analysis of the plaque constituents. OMSB was used to compare atherosclerotic plaques in proximal and distal regions of BCA in apoE/LDLR-/- mice. In conclusion, OMSB staining represents a novel staining that could be routinely used for qualitative and quantitative microscopic assessments of formaldehyde-fixed and paraffin-embedded sections of arteries with atherosclerotic lesions.

Prothionamide susceptibility testing of Mycobacterium tuberculosis using the resazurin microtitre assay and the BACTECMGIT 960 system.

Resazurin microtitre assay (RMA) has been successfully used to detect minimal inhibitory concentrations (MICs) of both first-line and several second-line drugs in drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB). In this study, we firstly compared prothionamide (PTH) susceptibility testing of Mycobacterium tuberculosis (MTB) using resazurin microtitre assay (RMA) and MGIT. Overall, the sensitivity and specificity of RMA for detecting PTH susceptibility was 96.5% [95% confidence interval (CI): 91.7-100.0] and 93.2% (95% CI: 89.6-96.8) respectively. In addition, the median time to positivity was significantly shorter for RMA than for the automated MGIT 960 (RMA, 8 days [range: 8-8 days] vs MGIT, 10.1 days, [range: 5.0-13.0]; P < 0.01). Concordance rate for MICs between RMA and MGIT for PTH-resistant group was 64.3% (95% CI: 46.5-82.0), which was significantly lower than that of PTH-susceptible group (85.9%, 95% CI: 78.8-93.0; P= 0.01). In conclusion, our data demonstrated that RMA can be used as an acceptable alternative for determination of PTH susceptibility with shorter turn-around time. When compared with MGIT 960, RMA method was prone to produce higher MICs for PTH-resistant MTB strains.

A fluorescence-activated cell sorting-based strategy for rapid isolation of high-lipid Chlamydomonas mutants.

There is significant interest in farming algae for the direct production of biofuels and valuable lipids. Chlamydomonas reinhardtii is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants of this organism with a perturbed lipid content are slow and tedious. Here, we present the Chlamydomonas high-lipid sorting (CHiLiS) strategy, which enables enrichment of high-lipid mutants by fluorescence-activated cell sorting (FACS) of pooled mutants stained with the lipid-sensitive dye Nile Red. This method only takes 5 weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantification of lipid content while preserving cell viability. We improved separation of high-lipid mutants from the wild type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20-fold enrichment of the known high-lipid mutant sta1 from a mixture of sta1 and wild-type cells. We then applied CHiLiS to sort thousands of high-lipid cells from a pool of about 60,000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that about 50% showed a reproducible high-lipid phenotype. We further characterized nine of the mutants with the highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had a higher triacylglycerol content and perturbed whole-cell fatty acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of CHiLiS opens the door to a systems-level understanding of green algal lipid biology by enabling genome-saturating isolation of mutants in key genes.

Broad-Specificity Chemiluminescence Enzyme Immunoassay for (Fluoro)quinolones: Hapten Design and Molecular Modeling Study of Antibody Recognition.

On the basis of the structural features of (fluoro)quinolones (FQs), pazufloxacin was first used as a generic immunizing hapten to raise a broad-specificity antibody. The obtained polyclonal antibody exhibited broad cross-reactivity ranging from 5.19% to 478.77% with 21 FQs. Furthermore, the antibody was able to recognize these FQs below their maximum residue limits (MRLs) in an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA), with the limit of detection (LOD) ranging from 0.10 to 33.83 ng/mL. For simply pretreated milk samples with spiked FQs, the ic-CLEIA exhibited an excellent recovery with a range of 84.6-106.9% and an acceptable coefficient of variation below 15%, suggesting its suitability and reliability for the use of a promising tool to detect FQs. Meanwhile, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models, with statistically significant correlation coefficients (q(2)CoMFA = 0.559, r(2)CoMFA = 0.999; q(2)CoMSIA = 0.559, r(2)CoMSIA = 0.994), were established to investigate the antibody recognition mechanism. These two models revealed that in the antibody, the active cavity binding FQs' 7-position substituents worked together with another cavity (binding FQs' 1-position groups) to crucially endow the high cross-reactivity. This investigation will be significant for better exploring the recognition mechanism and for designing new haptens.

Delamanid susceptibility testing of Mycobacterium tuberculosis using the resazurin microtitre assay and the BACTEC™ MGIT™ 960 system.

OBJECTIVES: The objective of this study was to develop standardized protocols for rapid delamanid drug susceptibility testing (DST) using the colorimetric resazurin microtitre assay (REMA) and semi-automated BACTEC™ MGIT™ 960 system (MGIT) by establishing breakpoints that accurately discriminate between susceptibility and resistance of Mycobacterium tuberculosis to delamanid. METHODS: MICs of delamanid were determined by the MGIT, the REMA and the solid agar method for 19 pre-characterized strains. The MIC distribution of delamanid was then established for a panel of clinical strains never exposed to the drug and characterized by different geographical origins and susceptibility patterns. WGS was used to investigate genetic polymorphisms in five genes (ddn, fgd1, fbiA, fbiB and fbiC) involved in intracellular delamanid activation. RESULTS: We demonstrated that the REMA and MGIT can both be used for the rapid and accurate determination of delamanid MIC, showing excellent concordance with the solid agar reference method, as well as high reproducibility and repeatability. We propose the tentative breakpoint of 0.125 mg/L for the REMA and MGIT, allowing reliable discrimination between M. tuberculosis susceptible and resistant to delamanid. Stop codon mutations in ddn (Trp-88 → STOP) and fbiA (Lys-250 → STOP) have only been observed in strains resistant to delamanid. CONCLUSIONS: We established protocols for DST of delamanid in the MGIT and REMA, confirming their feasibility in routine TB diagnostics, utilizing the same discriminative concentration for both methods. Moreover, taking advantage of WGS analysis, we identified polymorphisms potentially associated with resistance in two genes involved in delamanid activation.