Rapid colorimetric polymyxin B microelution directly from positive blood bottles: because patients with serious infections should not have to wait for results of culture-based methodologies.

PURPOSE: To evaluate the performance of the rapid colorimetric polymyxin B microelution (RCPEm) in determining polymyxin B resistance directly from Enterobacterales-positive blood cultures. METHODS: A set volume of positive blood culture bottles (diluted 1:10) was inoculated into a glucose-broth-phenol red solution (NP solution), where a polymyxin B disk was previously eluted (final concentration of 3 µg/mL). Test was read each 1 h for up to 4 h. Color change from red/orange to yellow indicated resistant isolates. Results were compared to the reference method, broth microdilution (BMD), performed from colonies grown on solid media from the same blood culture bottle. RESULTS: One hundred fifty-two Enterobacterales-positive blood cultures were evaluated, 22.4% (34/152) of them resistant to polymyxin B (including 6.6% with borderline MICs). When performing directly from positive blood cultures (RCPEm-BC), specificity and sensitivity were 99.1% and 94.1%, respectively. Of note, 79.4% (27/34) of truly resistant isolates required 3 h of incubation, compared to the 18 ± 2 h incubation that microtiter plates of BMD demand before reading can be performed. CONCLUSIONS: RCPEm directly from blood cultures has great potential to be part of the routine of clinical microbiology laboratories to establish polymyxin B susceptibility, impacting outcome of patients with bloodstream infections caused by carbapenem-resistant Enterobacterales.

Detection of polymyxins resistance among Enterobacterales: evaluation of available methods and proposal of a new rapid and feasible methodology.

BACKGROUND: Fast and accurate detection of polymyxins resistance is necessary as they remain the last resources to treat infections caused by Carbapenem-resistant Enterobacterales in many regions. We evaluated the rapid colorimetric polymyxin B elution (RCPE) and developed its miniaturized version, RCPE microelution (RCPEm), aiming to detect polymyxins resistance among Enterobacterales. METHODS: The methodologies consist of exposing the bacterial population in a solution (NP solution) where polymyxin B disks were previously eluted to obtain a concentration of 2 µg/mL for RCPE and 3 µg/mL for RCPEm. RESULTS: Two hundred sixty-seven Enterobacterales were evaluated, 90 (33.7%) resistant to polymyxin B by broth microdilution. It was observed 0.6% of major error (ME) by RCPE, with a specificity of 99.4%. The miniaturized version (RCPEm) presented the same ME and specificity values, but slightly higher sensitivity (97.8% vs. 95.6%) with 2.2% of very major error (VME). CONCLUSIONS: RCPE and RCPEm proved to be useful alternatives to determine polymyxin B susceptibility in clinical microbiology laboratories, presenting low cost, being easy to perform, and demanding short incubation time.

Validation of an Eco-Friendly Automated Method for the Determination of Glucose and Fructose in Wines.

Fermentable sugar dosage helps oenologists to establish a harvest's moment and control the fermentation process of the musts. The official analyses recommended for their determination are long, laborious, and must be carried out by specialized personnel. On the contrary, instrumental analysis automation limits human errors, increases precision, and reduces the time and cost of the analyses. In the food production sector, to use methods other than those recommended by supranational bodies in official reports, it is necessary to validate the analytical processes to establish the conformity of the results between the new methods and the reference ones. This work validated an automated enzymatic apparatus to determine the sum of glucose and fructose levels in wine samples. The validation was carried out on wine samples (dry red wine, dry white wine, moderately sweet wine, and sweet wine) containing different sugar concentrations by comparing data obtained using the OIV-MA-AS311-02 method performed by a specialized operator (reference method) and the same method performed by an automated apparatus. The difference between the results' means obtained with the two procedures was significant. Nevertheless, the automated procedure was considered suitable for the intended use since the differences between the averages were lower than the measurement uncertainty at the same concentration, and the repeatability results were better for the automated procedure than the reference method.

Peptide Amphiphile Mediated Co-assembly for Nanoplasmonic Sensing.

Aromatic interactions are commonly involved in the assembly of naturally occurring building blocks, and these interactions can be replicated in an artificial setting to produce functional materials. Here we describe a colorimetric biosensor using co-assembly experiments with plasmonic gold and surfactant-like peptides (SLPs) spanning a wide range of aromatic residues, polar stretches, and interfacial affinities. The SLPs programmed in DDD-(ZZ)x -FFPC self-assemble into higher-order structures in response to a protease and subsequently modulate the colloidal dispersity of gold leading to a colorimetric readout. Results show the strong aggregation propensity of the FFPC tail without polar DDD head. The SLPs were specific to the target protease, i.e., Mpro , a biomarker for SARS-CoV-2. This system is a simple and visual tool that senses Mpro in phosphate buffer, exhaled breath condensate, and saliva with detection limits of 15.7, 20.8, and 26.1 nM, respectively. These results may have value in designing other protease testing methods.

Development of RNA-Based Assay for Rapid Detection of SARS-CoV-2 in Clinical Samples.

INTRODUCTION: The ongoing spread of pandemic coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of growing concern. Rapid diagnosis and management of SARS-CoV-2 are crucial for controlling the outbreak in the community. Here, we report the development of a first rapid-colorimetric assay capable of detecting SARS-CoV-2 in the human nasopharyngeal RNA sample in less than 30 min. METHOD: We utilized a nanomaterial-based optical sensing platform to detect RNA-dependent RNA polymerase gene of SARS-CoV-2, where the formation of oligo probe-target hybrid led to salt-induced aggregation and change in gold-colloid color from pink to blue visibility range. Accordingly, we found a change in colloid color from pink to blue in assay containing nasopharyngeal RNA sample from the subject with clinically diagnosed COVID-19. The colloid retained pink color when the test includes samples from COVID-19 negative subjects or human papillomavirus-infected women. RESULTS: The results were validated using nasopharyngeal RNA samples from positive COVID-19 subjects (n = 136). Using real-time polymerase chain reaction as gold standard, the assay was found to have 85.29% sensitivity and 94.12% specificity. The optimized method has detection limit as little as 0.5 ng of SARS-CoV-2 RNA. CONCLUSION: We found that the developed assay rapidly detects SARS-CoV-2 RNA in clinical samples in a cost-effective manner and would be useful in pandemic management by facilitating mass screening.

A colorimetric test for the evaluation of the insecticide content of LLINs used on Bioko Island, Equatorial Guinea.

BACKGROUND: Insecticide-treated nets and indoor residual spraying of insecticides are used as the vector control interventions in the fight against malaria. Measuring the actual amount of deposits of insecticides on bed nets and walls is essential for evaluating the quality and effectiveness of the intervention. A colorimetric "Test Kit" designed for use as a screening tool, able to detect the type II pyrethroids on fabrics and sprayed walls, was used for the first time to detect deltamethrin on long-lasting insecticidal nets (LLINs) deployed on Bioko Island, Equatorial Guinea. METHODS: LLINs were analysed using the colorimetric Test Kit performed in situ, which leads to the formation of an orange-red solution whose depth of colour indicates the amount of type II pyrethroid on the net. The kit results were validated by measuring the amount of extracted insecticide using high-performance liquid chromatography (HPLC) with diode array detection (DAD). RESULTS: Deltamethrin concentration was determined for 130 LLINs by HPLC-DAD. The deltamethrin concentration of these nets exhibited a significant decrease with the age of the net from 65 mg/m2 (< 12 months of use) to 31 mg/m2 (> 48 months; p < 0.001). Overall, 18% of the nets being used in households had < 15 mg/m2 of deltamethrin, thus falling into the "Fail" category as assessed by the colorimetric Test Kit. This was supported by determining the bio-efficacy of the nets using the WHO recommended cone bioassays. The Test Kit was field evaluated in situ and found to be rapid, accurate, and easy to use by people without laboratory training. The Test Kit was shown to have a reliable linear relationship between the depth of colour produced and deltamethrin concentration (R2 = 0.9135). CONCLUSION: This study shows that this colorimetric test was a reliable method to assess the insecticidal content of LLINs under operational conditions. The Test Kit provides immediate results and offers a rapid, inexpensive, field-friendly alternative to the complicated and costly methods such as HPLC and WHO cone bioassays which also need specialist staff. Thus, enabling National Malaria Control Programmes to gain access to effective and affordable monitoring tools for use in situ.

Label-Free Pathogen Detection by a Deoxyribozyme Cascade with Visual Signal Readout.

A colorimetric nucleic acid based test for label-free pathogen detection has been developed and used for the detection of the Zika virus. The test relies on nucleic acid sequence-based amplification (NASBA) of a viral RNA followed by interrogation of the amplicon by a cascade of deoxyribozymes constituting a visual split deoxyribozyme (vsDz) probe. The probe consists of a split phosphodiesterase deoxyribozyme, which forms its catalytic core upon binding to a specific amplicon fragment. The catalytically active complex recognizes and cleaves an inhibited peroxidase-like deoxyribozyme (PDz), thereby activating it. Active PDz catalyzes hydrogen peroxide-mediated oxidation of a colorless substrate into a colored product, thereby generating a visible signal. Viral RNA (106 copies/mL or higher) triggers intense color within 2 hr. The test selectively differentiates between Zika and closely related dengue and West Nile viruses. The reported technology combines isothermal amplification and visual detection and therefore represents a basis for the future development of a cost-efficient and instrument-free method for point-of-care nucleic acid analysis.

Recent Advances in Colorimetric Tests for the Detection of Infectious Diseases and Antimicrobial Resistance.

Diagnosis of infection-causing microorganisms with sensitive, rapid, selective and economical diagnostic tests is critical to start the right treatment. With these tests, the spread of infections can be prevented. In addition to that, the detection of antimicrobial resistance also makes a significant contribution to public health. In recent years, different types of diagnostic tests have been developed as alternatives to traditional diagnostic tests used in clinics. In particular, colorimetric tests, which minimize the need for an instrument, have advantages owing to their cost effectiveness, rapid response and naked-eye detection and practical use. In this review, we especially focused on pH indicators and nanomaterial-based colorimetric tests in detection of infection-causing microorganisms and antimicrobial resistance.

Molecular test for COVID-19 diagnosis based on a colorimetric genomagnetic assay.

The world is in a long pandemic period caused by the SARS-CoV-2 virus and massive diagnostic tests to assist efforts to control the spread of the disease and also to avoid new coronavirus variants are still needed. Herein, we propose a simple and accurate saliva-based colorimetric test for the diagnosis of COVID-19. Magnetic beads (MBs) modified with a sequence of single-strand DNA (ssDNA) complementary to the N gene of the SARS-CoV-2 RNA were developed and used for magnetic capture and separation from a complex saliva sample. A second biotinylated ssDNA sequence was applied, and the colorimetric detection was carried out by adding streptavidin-horseradish peroxidase conjugate, H2O2, and tetramethylbenzidine (TMB) as chromogenic substrate. The test does not require viral RNA isolation, transcription, or amplification steps and can be performed at room temperature. The molecular assay test can be run using 96-well microplates, allowing the diagnosis of a large number of samples in 90 min. A simple support for magnets was designed and constructed using a 3D printer that allows the magnetic separations directly in the 96-well microplate. The colorimetric test showed an excellent ability to discriminate between healthy individuals and patients infected with SARS-CoV-2, with 92% and 100% of clinical sensitivity and specificity, respectively. This performance was similar to that achieved using the gold standard RT-PCR technique. The proposed genomagnetic assay offers an opportunity to greatly increase population testing, contribute to controlling the spread of the virus, and improve health equity in testing for COVID-19.

Chemical identification and optimization of the 4-aminophenol colorimetric test for the differentiation between hemp-type and marijuana-type cannabis plant samples.

The 4- Aminophenol (4-AP) colorimetric test is a fast, easy-to-use, and cost-effective presumptive assay of cannabis plant material producing different chromophores with THC-rich cannabis (blue color) and with CBD-rich cannabis (pink color). The main drawback of the 4-AP test is a brief observation window where the color rapidly changes to black, limiting the utility of the test. We now report for the first time, the identification of the product chromophores between 4-AP and CBD/THC as well as propose an explanation and a solution for the color degradation of the chromophores. The identification of the chromophores is provided by spectroscopic (UV-Vis), chromatography, and mass spectrometry (TLC and LC-QToF-MS). Oxidation of excess 4-AP (Reagent A) in the presence of NaOH (Reagent B) produces the black color observed for the previously reported 4-AP tests and reported in the literature. The adjustment of reactants concentrations and volumes of 4-AP:THC/CBD to a 1:1 ratio significantly reduces the black oxidation by-product and increases the observation window up to 2 h instead of the previously reported 5-10 min. For the first time, mass spectrometry and chromatography confirmed that the reaction of THC and CBD with 4-AP produced chromophores with m/z (M + H) = 420, consistent with proposed indophenol structures. The TLC method developed confirmed the separation between CBD and THC chromophores. The specificity of the test is also reported, showing false positive results for the presence of THC (blue color) for samples of thyme and oregano. LDA and SIMCA models showed that the optimized 4-AP procedure performs better than the previously reported 4-AP color test.

Resazurin microplate test method for rapid determination of colistin resistance in carbapenem-resistant Acinetobacter baumanii isolates.

BACKGROUND: Colistin-resistant Acinetobacter baumannii isolates are extremely important pathogens for hospital-acquired infections. OBJECTIVE: To investigate the effectiveness of the resazurin microplate assay (REMA) for the rapid determination of colistin resistance. METHODS: Susceptibility for colistin was investigated in vitro by the broth microdilution method (BMD) and the resazurin microplate assay (REMA) on 106 carbapenem-resistant Acinetobacter baumannii isolates. RESULTS: The results of both test methods were compared, and the categorical agreement between them was found to be 100%. No minor, major, or very major discrepancy was observed between the 2 methods. CONCLUSIONS: The most important advantages of REMA are that the results are obtained within 6 hours compared to the reference method, that it is easy to evaluate because it is colorimetric, and that the susceptibility result can be reported to the clinician on the same day as bacterial identification.

Highly sensitive fluorescent sensor for hypochlorite in nearly 100% aqueous solution and its application for live-cell, plant and zebrafish imaging.

A new ICT type D-π-A structured chemosensor DTB derived from a bithiophene-benzothiazole derivative has been synthesized. Sensor DTB showed a colorimetric and fluorometric dual-signaling response to hypochlorite (ClO-) in EtOH/HEPES solution (1/99, V/V, pH = 7.4, nearly 100% aqueous solution). Sensor DTB exhibited well specificity, high sensitivity and rapidity (<1 min) for ClO- with a detection limit of 25 nM. Sensor DTB features remarkable color changes and significant fluorescence "turn-on" response (ca. 45 fold) after treating with ClO-. Comprehensive analyses by 1H NMR, TLC, FTIR, HRMS, UV-vis, fluorescence and DFT illustrated that ClO- reacted with the CC bond of DTB, generating fluorophore 2T-CHO, leading to strong blue fluorescence. Interestingly, DTB loaded colorimetric test strips were established for rapid and real-time visual detection of ClO-. Furthermore, the DTB was successfully applied to quantitatively and sensitively detect ClO- in 84 disinfectant, bio-fluids (human serum and urine) and actual water samples. Importantly, the biocompatible DTB has been employed for visualizing and bioimaging ClO- in mung bean sprouts, Arabidopsis, live cells and zebrafish. These investigations demonstrate that DTB has great potentials for detecting ClO- in various biosystems and environments. This work would offer a new idea for developing multifunctional sensors with better performance for chemo/biosensors.

From a Chemotherapeutic Drug to a High-Performance Nanocatalyst: A Fast Colorimetric Test for Cisplatin Detection at ppb Level.

A rapid point-of-care method for the colorimetric detection of cisplatin was developed, exploiting the efficient conversion of the chemotherapeutic drug into a high-performance nanocatalyst with peroxidase enzyme mimics. This assay provides high specificity and ppb-detection sensitivity with the naked eye or a smartphone-based readout, outperforming many standard laboratory-based techniques. The nanocatalyst-enabled colorimetric assay can be integrated with machine-learning methods, providing accurate quantitative measurements. Such a combined approach opens interesting perspectives for the on-site monitoring of both chemotherapeutic patients to achieve optimal treatments and healthcare workers to prevent their unsafe exposure.

[Improvement Method of Confirmation Test of the In-pharmacy Formulation as Topical Medicine No. 37-①].

Topical Medicine No. 37-① is one of the in-pharmacy formulation that specifies a confirmation test (referred hereafter as the conventional method) to identify its ingredient diphenhydramine (DH) by colorimetric test. However, the conventional method is environmentally unfriendly due to the large amount of sample and organic solvent used, and the extraction process is complicated. We therefore developed three methods in view of improving the currently confirmation testing process: a simplified version of the conventional method, a TLC method, and an LC/photodiode array detector (PDA) method. The LC/PDA method was also examined for the quantitation of DH in order to determine its content in the medicine when needed. The LC/PDA method also demonstrated sufficient linearity in the calibration curve and recovery rate. We also evaluated whether the three methods developed in this study could be applied to Topical Medicine No. 37-①, which is made of absorptive cream containing different parabens.

Paper-Based Multiplexed Colorimetric Device for the Simultaneous Detection of Salivary Biomarkers.

In this study, we describe a monolithic and fully integrated paper-based device for the simultaneous detection of three prognostic biomarkers in saliva. The pattern of the proposed multiplexed device is designed with a central sample deposition zone and three identical arms, each containing a pre-treatment and test zone. Its one-step fabrication is realized by CO2 laser cutting, providing remarkable parallelization and rapidity (ca. 5 s/device). The colorimetric detection is based on the sensitive and selective target-induced reshaping of plasmonic multibranched gold nanoparticles, which exhibit a clear spectral shift (and blue-to-pink color change) in case of non-physiological concentrations of the three salivary biomarkers. A rapid and multiplexed naked-eye or smartphone-based readout of the colorimetric response is achieved within 10 min. A prototype kit for POCT testing is also reported, providing robustness and easy handling of the device.

Nanosensors for Visual Detection of Glucose in Biofluids: Are We Ready for Instrument-Free Home-Testing?

Making frequent large-scale screenings for several diseases economically affordable would represent a real breakthrough in healthcare. One of the most promising routes to pursue such an objective is developing rapid, non-invasive, and cost-effective home-testing devices. As a first step toward a diagnostic revolution, glycemia self-monitoring represents a solid base to start exploring new diagnostic strategies. Glucose self-monitoring is improving people's life quality in recent years; however, current approaches still present vast room for improvement. In most cases, they still involve invasive sampling processes (i.e., finger-prick), quite discomforting for frequent measurements, or implantable devices which are costly and commonly dedicated to selected chronic patients, thus precluding large-scale monitoring. Thanks to their unique physicochemical properties, nanoparticles hold great promises for the development of rapid colorimetric devices. Here, we overview and analyze the main instrument-free nanosensing strategies reported so far for glucose detection, highlighting their advantages/disadvantages in view of their implementation as cost-effective rapid home-testing devices, including the potential use of alternative non-invasive biofluids as samples sources.

Colorimetric Point-of-Care Detection of Clostridium tyrobutyricum Spores in Milk Samples.

Clostridium tyrobutyricum represents the main spoiling agent responsible for late blowing defects (LBD) in hard and semi-hard cheeses. Its spores are resistant to manufacturing procedures and can germinate during the long ripening process, causing the burst of the cheese paste with a consequent undesirable taste. The lower quality of blown cheeses leads to considerable financial losses for the producers. The early identification of spore contaminations in raw milk samples thus assumes a pivotal role in industrial quality control. Herein, we developed a point of care (POC) testing method for the sensitive detection of C. tyrobutyricum in milk samples, combining fast DNA extraction (with no purification steps) with a robust colorimetric loop-mediated isothermal amplification (LAMP) technique. Our approach allows for the sensitive and specific detection of C. tyrobutyricum spores (limit of detection, LoD: ~2 spores/mL), with the advantage of a clear naked-eye visualization of the results and a potential semi-quantitative discrimination of the contamination level. In addition, we demonstrated the feasibility of this strategy using a portable battery-operated device that allowed both DNA extraction and amplification steps, proving its potential for on-site quality control applications without the requirement of sophisticated instrumentation and trained personnel.

Evaluation of CARBA PAcE, a novel rapid test for detection of carbapenemase-producing Enterobacterales.

Introduction. Carbapenemase-producing Enterobacterales (CPE) are an increasing threat to global health. Fast detection is crucial for patient management and outbreak control.Hypothesis/Gap statement. Recently, a new commercial colorimetric test, CARBA PAcE, was released that has not yet been scientifically evaluated.Aim. Our goals were to evaluate the performance of CARBA PAcE using a large variety of different CPE.Methodology. CARBA PAcE was challenged with 107 molecularly characterized CPE and 53 non-CPE controls. Isolates were grown on Mueller-Hinton agar (MHA); in the case of a false-negative result, isolates were additionally inoculated on Columbia blood agar (CBA) and CARBA PAcE was repeated. The test was performed according to the manufacturer's protocol.Results. CARBA PAcE showed an overall sensitivity and specificity of 72 % [confidence interval (CI) 62-80 %] and 91 % (CI 79-97 %), respectively, when isolates were grown on MHA. With growth on CBA, detection improved (especially of metallo-ß-lactamases), resulting in an extrapolated sensitivity of 89 % (CI 81-94 %) for all carbapenemases and 96 % (CI 89-99 %) for the four major carbapenemases (NDM, OXA-48-like, KPC, VIM).Conclusion. CARBA PAcE is a simple and very rapid test for the detection of CPE which performs well for the major carbapenemases when isolates are grown on CBA. Laboratories should be aware of the limitations of this assay, such as moderate sensitivity when isolates are grown on more challenging agars such as MHA and the poor detection of some rare carbapenemases (e.g. IMI, OXA-58).

Drug Checking as Strategy for Harm Reduction in Recreational Contests: Evaluation of Two Different Drug Analysis Methodologies.

Introduction: Drug checking as a part of drug harm-reduction strategies represents an essential aspect of public health policies. It focuses on rapid identification of drugs that individuals intend to use during night events, in order to implement health-protective behaviors. Chemical drug analysis techniques vary considerably, from simple colorimetric reagents to advanced forensic methods such as gas chromatography/mass spectrometry (GC/MS). Materials and Methods: In 2019, drug-check services were offered at some night events in Umbria (Central Italy). One hundred and twenty attendees directly delivered unidentified substances to a harm-reduction worker, who collected a few milligrams of the substances on ceramic plates and added a drop of colorimetric reagent. Multiple reagents were used to increase the diagnostic capacity of a substance, which may react with a specific drug or a few drugs. Later, a fraction of the samples was analyzed by GC/MS. The concordance of the results obtained using these two methodologies and the intended behaviors of consumers after being informed of the test result was evaluated. Results: We analyzed 120 samples by colorimetric test: 32 MDMA, 25 ketamine, 10 amphetamine, 11 cocaine, 8 heroin, and 4 LSD samples. The results were inconclusive for 29 samples. The GS/MS analysis confirmed MDMA in 84%, ketamine in 78%, amphetamine in 91%, cocaine in 92%, heroin in 88%, and LSD in 100% of the samples. The results of samples with inconclusive results were as follows: 2, MDMA; 7, ketamine; 2, amphetamine; 2, cocaine; 2, heroin; 2, mephedrone; 6, mixes; 1, debris; and 5, adulterants as the main component. Twenty-one of 29 participants reported that they had no intention of consuming the unidentified substance. Discussion: The high percentage of individuals who claimed no intention of consuming the unidentified drugs indicates that drug checking is viable as a part of drug harm-reduction strategies. Overall, colorimetric reagents showed a good performance with regard to samples being unadulterated (LSD) or minimal in quantity, but failed to identify mixtures of substances and the adulterants present in them. Therefore, the use of more discriminatory on-site methods such as Raman or infrared spectrometry is strongly recommended.

A Rapid Colorimetric Assay for On-Site Authentication of Cephalopod Species.

A colorimetric assay, exploiting the combination of loop-mediated isothermal amplification (LAMP) with DNA barcoding, was developed to address the authentication of some cephalopod species, a relevant group in the context of seafood traceability, due to the intensive processing from the fishing sites to the shelf. The discriminating strategy relies on accurate design of species-specific LAMP primers within the conventional 5' end of the mitochondrial COI DNA barcode region and allows for the identification of Loligo vulgaris among two closely related and less valuable species. The assay, coupled to rapid genomic DNA extraction, is suitable for large-scale screenings and on-site applications due to its easy procedures, with fast (30 min) and visual readout.