IT202200000689A1 - USE OF DYES IN THE SELECTIVE IDENTIFICATION OF INFECTED SITES IN BIOFILM-RELATED INFECTIONS - Google Patents

Description

PATENT FOR UTILITY MODEL?

TITLE: USE OF DYES IN THE SELECTIVE IDENTIFICATION OF INFECTED SITES IN BIOFILM-RELATED INFECTIONS

DESCRIPTION

1-TECHNICAL FIELD

The present invention concerns the use of dyes, their mixtures and metal complexes obtained from them, in the form of a solution, suspension, emulsion or spray, for the selective identification of infected sites during the surgical treatment of osteomyelitis, skin infections and soft tissues and peri-prosthetic and fixation infections.

2-STATE OF THE ART

Almost all of microbial infections in the human body are related to the formation of biofilm which significantly contributes to morbidity? and mortality?, especially in hospital environments Understanding biofilm resistance to antibacterial agents. Nat Rev Drug Discov. 2003; 2:114-122]. Many of these are infections related to the use of medical devices such as central venous catheters, urinary catheters, heart valves, joint prostheses, intrauterine devices, contact lenses, acrylic dental prostheses, on whose surface microbial populations can adhere. In particular, peri-prosthetic infections such as those of synthetic media represent one of the most great challenges from the diagnostic, surgical and post-operative management points of view in the field of orthopaedics, since? they are very often associated with morbidity? high, need? to prolong hospitalization and, therefore, to an increase in healthcare costs. Typically, the incidence of this complication varies from 0.5% to 2% of all prosthetic replacement operations [

(2010) The epidemiology of revision total knee arthroplasty in the United States. Clin Orthop Relat Res 468:45-51;

(2009) The epidemiology of revision total hip arthroplasty in the United States. J Bone Joint Surg Am 91:128-133]. Different ? the incidence of infections associated with osteosynthesis devices (intramedullary nails, plates, screws, wires, transcutaneous fixation pins) which varies from 1-2% for closed fractures to over 30% for open ones.

Regardless of the incidence, the difficult therapeutic management of patients suffering from this serious complication is related to the fact that biofilm-related infections are infections that affect the device (prosthetic implant or synthesis means), the peri-implant soft tissues including the skin and finally the bone (osteomyelitis). The bacteria that reach the surgical site can bind to various molecules, including fibrinogen, as well as extracellular matrix proteins (fibronectin and laminin) and glycosaminoglycans. These proteins not only allow surface colonization, but are also responsible for a reduction in local defense mechanisms. Once adhered, the bacteria multiply, forming macrocolonies and subsequently produce an extracellular matrix (slime). The biofilm represents a structure within which bacteria and fungi increase their resistance towards the host's immune system and antimicrobial agents. For these reasons, antibiotic therapy, unfortunately, often fails to eradicate and completely eliminate the biofilm and is for this reason, infections caused by biofilm-producing bacteria occur recurrently and insidiously, making it necessary to have adequate cycles of antibiotic therapy associated with surgical therapy which requires careful debridement and the removal of the prosthetic implant or of the fixation devices in the case of fractures.

The main pathogens responsible for prosthetic infections, for example but not only for hip and knee operations, are listed in table 1, TABLE I.

Bacteria and detection techniques

Bacteria make up the microbial ecosystem in and around our body.

Their classification? mainly based on differences in cellular composition, metabolism and cellular structure.

To date, the methods used for their identification and classification typically involve pre-enrichment, selective enrichment, biochemical screening and serological confirmation steps. These methods are certainly selective and specific, but, since? are based on a series of enrichment steps, the results are often difficult to interpret and not available on the desired time scale for rapid corrective action. For example, methods such as genetic analysis approaches, such as PCR (polymerase chain reaction), FISH (fluorescent in situ hybridization), and NGS (Next Generation Sequencing (NGS) or parallel sequencing) are powerful techniques for identifying bacteria but, due to the great preparatory work and the enormous amount of data necessary for an in-depth analysis, they are often inappropriate methods in clinical diagnosis, which instead requires rapidity. if not even in situ and real-time monitoring.

The methods for bacterial identification commonly used today are also typically characterized by poor sensitivity, and the rapid detection of a limited number of cells (<10 CFU/mL) is a challenge still open. Among the diagnostic methods that involve the use of dyes we remember:

Gram stain: Gram stain? the standard technique for bacterial classification. It is based on the different properties? physics of the bacterial cell wall structure. ? a rather consolidated and reliable technique and is been applied to various forms of bacterial specimens, in culture and in infected tissues.

However, Gram staining requires several steps, and in particular the fixation of the bacterial cell, two staining steps with two different dyes, a final decolorization and washing step. From the complexity? of the method derive some intrinsic limitations of the technique: the decolorization phase, for example, often generates a false determination of the Gram while the washing phase can? cause significant cell loss.

Assay using crystal violet (CV): staining using crystal violet (CV)? one of the first methods adopted for the quantification of bacterial biofilm biomass [

Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 1985; 2: 996-1006;

A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 2000; 40: 175-9]. This method consists of staining negatively charged molecules using hexamethylpararosaniline (CV) chloride. The limitations of this method are related to the poor reproducibility, related to the growth of the biofilm, the specific nature and concentration of the solvent used and the elution times. Furthermore, since? both living and dead cells, so? as the biofilm matrix are stained with CV, this method does not provide information on the actual number of living bacteria and therefore ? poorly suited for evaluating the anti-biofilm efficacy of antimicrobial substances.

Assay using 1,9-dimethyl methylene blue (DMMB): this method is based on the consideration that the cationic dye DMMB detects the intercellular polysaccharide adhesin (PIA) which represents one of the main constituents of the extracellular matrix that makes up the S. aureus biofilm. This method? easy to perform, economical and requires little execution time. The main limitation seems to be what ? limited to the identification of those few bacterial species such as Staphylococcus aureus in which this molecule is present in the biofilm matrix.

Assay using fluorescein-di-acetate (FDA): this technique involves the use of fluorescein-di-acetate (FDA), which represents a soluble dye and for these reasons penetrates the cell membrane. After bacterial absorption, the FDA ? hydrolysed by bacterial esterase producing florescin which emits yellow fluorescence whose signal can be quantified using a spectrophotometer. Although this method is easy to perform and inexpensive, it is not? widely used as it is not ? particularly suitable for quantitative studies on mature biofilm, producing only semi-quantitative results [

Comparison of three assays for the quantification of Candida biomass in suspension and CDC reactor grown biofilms. J Microbiol Methods 2005; 63: 287-95].

Live/Dead Dye Assay: This method is based on the use of two different nucleic acid dyes that differ in their ability to dye. to penetrate bacterial cells. The first dye ?SYTO 9 green fluorescent nucleic acid stain? ? a commercial product capable of crossing all bacterial membranes and binding to DNA in both Gram-positive and Gram-negative bacteria. The second dye? red fluorescent (propidium iodide) that passes through damaged bacterial membranes. In this way, bacteria with an intact plasma membrane (identified as viable), stained with SYTO 9, emit green fluorescence, while those with a damaged membrane, stained with PI, are colored red.

Recently, several antibiotic-derived antibiotics have also been reported as new bacterial probes, but their activity? biological can? interfere with bacterial function along with the potential problem of antimicrobial resistance.

An ideal probe should be selective for all Gram-positive and Gram-negative bacteria without any biological interference and should not require any washing or decolorization steps to avoid complications in sample handling and data analysis.

Likewise, in recent years yes? witnessed a dramatic increase in resistant bacteria in hospitals and healthcare facilities in general, due to the excessive use of antibiotics and careless, ineffective or inadequate cleaning also due to the lack of a rapid and inexpensive test to verify the ?effective sanitization of surfaces and/or equipment and tools used. Finally, even in the food processing sector, the detection of harmful levels of microbes? very important to ensure quality? of the product to the final consumer. Again, in order to ensure the safety of the food supply, fast and economical monitoring of microbe levels in the food processing industry ? fundamental.

Dyes and metal nanoparticles

Dyes are organic molecules characterized by the presence of a group, called chromophore, which gives the dye its characteristic properties. of light absorption. Chromophores are generally represented by aromatic rings and/or chains of carbon atoms linked by double bonds, conjugated to each other, in order to guarantee a delocalization of electrons. The position and composition of the atoms or groups of atoms influence the coloration of the chromophore and characterize each class of dyes. Examples of dye classes include

i) Carotenoids which have a variable color between yellow and red, such as carotene, retinol, vitamin A, zeaxanthin;

ii) quinones, whose color ranges from yellow to red-violet, such as carminic acid, extracted from cochineals, which is used as a textile dye;

iii) tetrapyrrole dyes, characterized by ranging from red to blue-green and which include, among other things, chlorophyll, hemoglobin, the pigments of the respiratory chain and vitamin B12;

iv) pyran dyes, with variable colouring; these include anthocyanins, blue-red violet, responsible for the color of many flowers and fruits; flavones, varying from orange to yellow, which in plants selectively absorb certain wavelengths important for photosynthesis, while reflecting ultraviolet light that is harmful to cells.

Many dyes, such as fuchsin, eosin, methylene blue, aniline blue, perform important activities. biochemicals. Among these, the use of these molecules as antimicrobial agents or for the detection of bacteria is of particular interest.

Important feature of many dyes? that of easily coordinating with transition metals, through a particularly stable type of bond, called coordination bond. The establishment of this bond often confers properties new and interesting optics from a technological point of view to the related metal complexes, which therefore can also be usefully used in chemical diagnostics, instead of or in addition to the dyes from which they derive.

Nanoparticles of metals or inorganic compounds have been known for many years. A nanoparticle? a particle with dimensions in the range 1-100 nm. It presents peculiar properties, not found in the corresponding bulk material, nor? into particles having the same composition but characterized by larger dimensions.

The atypical surface structure, the very high surface area to volume ratio and the consequent greater reactivity make these particles useful in many commercial applications. In particular, nanoparticles characterized by a diameter smaller than ~30 nm are characterized by high surface energy and are thermodynamically unstable. In such cases, crystallographic changes have in fact been observed such as the expansion or contraction of the lattice, a change in morphology, the appearance of defects or the rearrangement of the atoms on the surface in an attempt to reduce the surface energy of the unstable particles.

Even some properties? physics of nanoparticles, such as their properties? electrical, the Curie temperature, or the melting temperature, can depend on their size and shape.

Of particular interest? the capacity? of nanoparticles to act as fluorescent probes for biology and the fact that gold, silver and copper nanoparticles exhibit surface plasmon resonance in the visible region of the electromagnetic spectrum, which gives? originates intensely colored materials that alter their color with the direction of observation. This ? a phenomenon known since ancient times, when the Romans produced glass objects such as the Lycurgus cup, which contained Ag/Au nanoparticles, and which made the object red or green depending on whether it was observed in transmitted or reflected light .

Surface plasmon resonance (SPR) is a phenomenon at the basis of many instruments for measuring the adsorption of material on planar metal surfaces (generally gold and silver) or on surfaces of metal nanoparticles, and it is the operating principle of many color-based biosensors.

The SPR of nanoparticles strongly depends on their size and shape. Therefore, for example, the color of a gold colloid can give a precise idea of the size of the nanoparticles it contains.

The SPR of a nanoparticle? also very sensitive to the nature of the molecules possibly coordinated with the metal. Therefore, chromophores covalently bound to the surface of nanoparticles can act as simple chemical sensors.

Synthesis of noble metal nanoparticles

There are many synthesis routes to obtain gold and silver nanoparticles, and the scientific literature on the subject? very rich. Among the many synthetic strategies, two general categories can be identified: top-down or bottom-up approaches.

Top-down methods include electron beam lithography and photolithography. These methods suffer from the obligation to remove large quantities. of material which is therefore wasted. Furthermore, they impose strict limits on the size of the nanostructures that can be generated, since photolithography? constrained by the diffraction limit of the available lasers. Does current technology allow you to generate functionality? in the range 20-100 nm, but? however constantly improving. Bottom-up techniques have attracted greater interest because are they more? versatile and require less specialized equipment. The general approach consists in using a reducing agent to reduce the metal ions in solution, thus determining the organization of the atoms into nanostructures. The techniques in this area are many and varied, including ?templating?, chemical, electro-photochemical and thermal reductions. Bottom-up methods allow very small nanostructures (from 1 nm) to be fabricated but require a stopping agent to stop the growth of the particles at the desired location. Bottom-up methods allow excellent control of the shape and size of the nanoparticles produced, through stringent control of reaction parameters such as time, temperature, nature of the reducing agent and arresting molecules. Although most nanoparticles obtained by such techniques are spherical, various reaction conditions and methods have produced nanoparticles in the form of wires, tubes, prisms, or tetrahedra. A negative aspect of bottom-up techniques? the extreme variability? of the synthesized nanoparticles, due to the extreme sensitivity? to fluctuations in temperature, pH, time etc.

Interaction of cationic and anionic dyes with gold and silver nanoparticles

Noble metal nanoparticles not only exhibit a number of properties rather unique chemical-physical properties, but they also have the ability? to influence the properties? of the molecules to which they can be coordinated. For example, the addition of a silver nanoparticle dye involves a significant modification of the UV-VIS spectrum compared to that of the pure dye.

Scientific literature has widely demonstrated that the coordination of dyes with nanomaterials can? create potent antimicrobial agents that can be used for medical purposes, both in photodynamic therapy and as antimicrobial surfaces.

Light-activated antimicrobial agents, such as toluidine blue O (TBO), are effective on a wide range of bacteria including methicillin-resistant Staphylococcus Aureus (MRSA). These reagents work by producing free radicals and reactive oxygen species when activated by white light or laser. Radicals are responsible for the destruction of bacteria through a multi-objective mechanism (unlike antibiotics which, on the contrary, are very specific) and this should minimize the development of acquired resistance. These new systems are particularly useful for topical infections, such as wounds and in the cavity. oral, and for this reason, in recent years, they have been at the center of intense scientific interest.

Of great interest are the water-soluble gold nanoparticles, functionalized with thiolate. One of the synthesis methods, the Brust method, is particularly interesting since it allows control over the size of the particles, their dispersion and the effectiveness of surface functionalization and also allows obtaining functionalized nanoparticles with properties size-dependent optical and catalytic. Furthermore, these nanoparticles are stable in air, and are preserved long-term in solvents, miscible with water, even at high temperatures and extreme concentrations.

Functionalized gold nanoparticles have found multiple applications in the biochemistry sector, as they ? Is it possible to modify them with simple methods to better adapt them to biological systems by modifying their surface layer for greater solubility? aqueous and biocompatibility.

Spectroscopic techniques for the identification of dyes and their complexes

Surface-amplified Raman spectroscopy (Surface Enhanced Raman Scattering, SERS) is a technique that allows you to increase sensitivity? of Raman spectroscopy of a factor that can? reach up to 1014-1015. This sensitivity? ? therefore sufficient to also allow the detection of single molecules. SERS spectroscopy? therefore interesting for the analysis of trace materials, flow cytometry and other applications where speed is needed. analysis and high sensitivity.

Does the SERS phenomenon take place on a metal surface that has roughness? on a nanometric scale and? therefore useful for the detection and analysis of molecules adsorbed on that surface. Typical metals used to create SERS-active surfaces are gold or silver: can surface preparation be done? occur via electrochemical roughening, metal coating of a nanostructured substrate, or deposition of metal nanoparticles (often in colloidal form).

SERS substrates can be used to detect the presence of biomolecules at low concentrations: of particular interest? the development of SERS tags for cancer detection, as well as? of SERS-labeled antibodies in SERS immunoassays for protein localization in plasma/serum or blood tissues. The capacity? The ability to analyze the composition of a mixture at the nanoscale also makes the use of SERS substrates advantageous for environmental analysis, pharmaceuticals, materials science, forensics, and single cell detection. The application of SERS techniques to biochemical diagnostics and future implementation in clinical practice? therefore a rather consolidated technique but which at the same time reserves wide margins for development [in Encyclopedia of Spectroscopy and Spectrometry (Third Edition), 2017]

Surgical treatment of biofilm-related infections Once the septic process has established, the possibilities? of treatment are multiple. In cases of chronic infection, surgical treatment involves the removal of the implant associated with careful debridement. As regards peri-prosthetic joint infections, the most suitable surgical treatment? effective? represented by the implantation of an antibiotic-containing cement spacer which has the purpose of reclaiming the infected site in anticipation of a subsequent intervention (two-stage technique) [

The Fate of Spacers in the Treatment of Periprosthetic Joint Infection.]. Although the role of careful debridement of all infected and necrotic tissue plays a key role in the treatment of an infection [Insall JN, Thompson FM, Brause BD. Two-stage reimplantation for the salvage of infected total knee arthroplasty. J Bone Joint Surg Am. 2002 Mar;84(3):490.

Methylene Blue Guided Debridement as an Intraoperative Adjunct for the Surgical Treatment of Periprosthetic Joint Infection J Arthroplasty. 2017 Dec;32(12):3718-3723.], the evaluation of the resection margins between healthy and pathological tissue is quite complex and often entrusted to the surgeon's experience with the increased risk of both inadvertent removal of the healthy tissue and failure to remove all pathological tissue.

3-DESCRIPTION OF THE INVENTION

The purpose? of this utility model? ? that of extending the use of dyes, mixtures and their nanometric complexes, already? extensively described in the scientific and patent literature, to the selective identification of infected sites during the surgical treatment of osteomyelitis and peri-prosthetic infections during surgery, with accurate identification of the resection margin between healthy and pathological tissue, so to be able to guide a selective and accurate debridement.

Currently, in clinical practice, staining techniques are typically used to label dried bacteria on slides, but not in fluids or in vivo. The same applies to the staining of bacterial biofilm which generally occurs in laboratory settings for purely experimental and non-clinical purposes.

By means of the present invention, gram-negative and grampositive bacteria as well how fungi can be marked for simple and rapid analysis. In this way the presence of bacteria can therefore be quickly and easily detected without resorting to microscopic examinations.

This invention? aimed at the development of a ready-to-use kit for the rapid detection of bacteria and bacterial and fungal biofilm. This device allows the doctor to determine (qualitatively) the type of infection and (quantitatively) its extension and responds to the current need? to have a rapid, objective, easy-to-use screening

The system in question is based on the use of a coloring system sensitive to microbes which undergoes a rapid, clearly visible and measurable color change in their presence. In the presence of microbes, the change must be visibly detectable to the naked eye, without the aid of additional instruments, or by UV-VIS spectrometry.

This system? based on a mixture developed by us of organic dyes, coordinated and non-coordinated with metal nanoparticles, for example but not limited to Au, Ag, Cu, Pt, Pd. The choice of the study of metal nanoparticles derives from their high capacity? of interaction with visible radiation which makes them particularly useful as chemical and biological sensors even capable of detecting a single target molecule. These substances can be considered chemically and biologically inert and, appropriately functionalized to recognize receptors present only in pathological tissues, they can accumulate in diseased tissues and carry out a diagnostic and indirectly therapeutic action.

This coloring system can be applied to fabrics and surfaces in general to reveal not only the presence of microbes but also their concentration. In fact, the intensity? of the coloring, what can? be easily measured through colorimetric measurement with fiber optic reflectance spectroscopy (FORS),? proportional to their concentration.

The coloring system can be in the form of a liquid, solid or gel, and preferentially of a liquid that can? be nebulized, sprayed on fabrics or surfaces to indicate the presence of microbes.

A further application provides that the liquid containing the coloring system can be applied to the surfaces and left to dry, forming an active film which, in the event of subsequent contamination by microbes, is able to reveal it, changing color. This method can be useful for use on surfaces such as, for example, packaging, stickers, paper, fabrics, worktops, medical accessories such as surgical gloves, surgical gowns and curtains, to make any contamination quickly and easily detectable.

According to the present invention the color change must occur rapidly: in some embodiments the coloring system can start changing color in less than 10 seconds, in others in about 30 minutes. In some embodiments, the dye changes color at a faster rate. proportional to the concentration of microbes. In other embodiments, the quantity of bacteria present? proportional to a quantity? of dye that undergoes a change.

3.1-INVENTION DETAILS

In the examples described below are the methods shown? with which two coloring mixtures were effectively used in the detection of bacteria on infected and non-infected tissues and devices, taken during surgery.

Example 1

? Preparation of the dye mixture

A 60 ml solution of dye is prepared by mixing: ? From 0 to 15 ml of aqueous solution of methylene blue at 0.0025% by weight; from 0 to 15 ml of Toluidine blue aqueous solution at 0.0025% by weight; from 0 to 15 ml of Fuchsin aqueous solution at 0.02% by weight; from 0 to 15 ml of Eosin aqueous solution at 0.01% by weight

Example 2

? Preparation of functionalized nanoparticles Silver or gold nanoparticles were prepared according to different methodologies in accordance with what is reported in the literature.

Silver nanoparticles can be prepared according to the following procedures, and more:

1) a sample of AgNO3 (about 90 mg)? was suspended in 500 mL of distilled water, and heated to 100?C under nitrogen flow; 10 mL of a 1% aqueous solution of sodium citrate were added to the suspension, drop by drop, under continuous stirring and under a nitrogen flow. The solution ? was kept boiling for 60-90 minutes. Is the brownish-yellow suspension so? obtained, which shows an absorption maximum at approximately 415 nm, ? made up of particles approximating spheres with an average diameter of about 20-40 nm.

2) A solution of 5x10-3 M AgNO3, (100 mL) ? was added slowly and under vigorous stirring to 300 mL of a 2x10-3M NaBH4 solution at approximately 0°C. During the reduction? 1% PVA solution (50 mL) was added. The mixture ? was then boiled for approx. 1h to decompose any excess NaBH4. The solution obtained showed an absorption maximum at 400 nm and ? made up of particles approximating spheres with an average diameter of about 20-40 nm.

3) A sample of AgSO4 (80 mg) ? was dissolved in 200 mL of water at 100?C and then mixed with 5 g of PVA dissolved in approx. 200 mL of water at 80?C. The mixture ? was then bubbled with H2 at near boiling temperature for 3 hours. The solution obtained showed an absorption maximum at 400 nm and ? made up of particles approximating spheres with an average diameter of about 20-40 nm.

Gold nanoparticles were prepared according to the following procedures:

1) 250 mg of HAuC14 were dissolved in 500 mL of ultrapure water and the solution ? was brought to the boil. Under agitation and in nitrogen flow, ? 1% sodium citrate solution (50 mL) was then slowly added. The boil? continued for approx. 1 hour. The solution obtained, red in colour, showed an absorption maximum at 530 nm and ? made up of particles approximating spheres with an average diameter of about 20-50 nm.

2) A solution of 5x10-3M HAuCl4 (100 mL) ? was added dropwise and under strong stirring to 300 mL of a 2x10-3M NaBH4 solution, kept at 0°C. During the reduction? 1% PVA solution (50 mL) was added. After completion of the reaction, the mixture is was boiled for 1 hour to decompose the excess NaBH4. The solution obtained, purple in colour, showed an absorption maximum at 530 nm and was made up of particles approximating spheres with an average diameter of approximately 20-50 nm.

The synthesis of functionalized nanoparticles? was carried out according to methodologies reported in the literature (see for example J. Am. Chem. Soc., 1999, 121, 7081; Mater. Chem., 2007, 17, 3739;

Carbohydrate Polymers 2012, 89, 1159,

Vol. 14, No. 26, 1998).

Complexation with dyes? was then carried out by adsorbing the dyes used (Methylene blue, Toluidine blue, Fuchsin, Eosin) onto the nanoparticles produced as described above, placing each suspension in contact with a solution of different concentration (10-3 -10-7 M) of the dye choice. The suspension ? was then kept under vigorous stirring for 90 minutes at 25°C.

Example 3

Target patients

Patients undergoing surgery for suspected or confirmed biofilm-related infection (infections of synthetic media, joint prostheses, osteomyelitis and chronic skin and soft tissue infections).

Tissue collection phase:

Pickup location

The withdrawal? was carried out according to aseptic standards, in a sterile field, using sterile material and with an operator assisted in opening the packages.

Mode? of the withdrawal

After preparing the skin with a solution based on chlorhexidine digluconate or povidone iodine, left to act for at least 2-3 minutes, we proceed with skin access to the limb, with the limb bloodless thanks to the use of ischemic bandage. At this point, access to the infected or suspected site is performed, subsequently carrying out extensive debridement, also avoiding the use of the electric scalpel in this case. The tissue sampling must also include, where possible, a large and evident portion of subcutaneous fat. The sample obtained, approximately 3 x 3 cm, is placed immediately in a sterile container. The same goes for the management of the explanted device which is inserted into a sterile container to reduce the possibility of damage. of bacterial contamination.

Tissue sample management.

The sample is transported in the shortest possible time (< 2 h) to an appropriate analysis laboratory. If this is not possible, the withdrawal will go? stored at a temperature of 4°C for a maximum of 12 hours.

Device management

The sample is transported in the shortest possible time (< 2 h) to an appropriate analysis laboratory. If this is not possible, the withdrawal will go? stored at a temperature of 4°C for a maximum of 12 hours.

Analysis phase:

Phases of the experiment? tissue sample

Once arrived in the destination laboratory, the tissue samples are freed from the attached blood clots by washing three times with sterile PBS (phosphate buffered saline, isotonic cell and buffered solution at pH 7.4 consisting of water and NaCl KCl Na2HPO4 KH2PO4) solution.

Subsequently the sample? placed in a sterile container to which 60 ml of dye solution is added using an antibacterial filter. The tissue sample must remain immersed for a maximum of 3 minutes and subsequently subjected to 3 washing cycles with PBS to eliminate excess dye. All the aforementioned phases must be carried out in conditions of absolute sterility.

Removal of colored portions.

The areas that change the most in the context of the sample are selected through inspection. markedly towards a dark purple color; from these, 3 samples of approximately 1 cm x 1 cm (proband tissue) are sterilely taken. Furthermore, 3 further areas which are not colored are selected, from which 3 further 1 x 1 cm samples will be taken (control tissue). All samples obtained will be subjected to microbiological examinations.

Phases of the experiment? Device

Once it arrives in the destination laboratory, the device is freed from the attached blood clots by washing three times with sterile physiological solution (NaCl 0.9%) and added to the coloring solution for a maximum of 3 minutes. The excess dye is then eliminated by subjecting the sample to 3 washing cycles with sterile physiological solution (NaCl 0.9%). All the aforementioned phases must be carried out in conditions of absolute sterility. The sample is subsequently subjected to microbiological tests.

Example 4.

The stained and unstained tissue samples are homogenized and added to 5 ml of enrichment medium (Brain-Heart) for 1 minute. For germ detection using traditional microbiology, approximately 0.5 ml of broth homogenate is inoculated into blood culture bottles for aerobic and anaerobic microorganisms and incubated for 14 days at 36? 1?C. These samples are subcultured to blood agar, chocolate agar and Schaedler agar when the broth becomes cloudy or at the end of incubation. Microbiological analysis using biochemical methods is used for all samples. Real-time PCR is performed using species-specific primers designed to assess bioburden. These techniques aim at the detection of a predetermined genus or species based on the clinical and epidemiological context.

Example 5.

The containers containing the device are opened under a hood and covered for at least 90% of its volume with Ringer's solution or sterile physiological solution. Proceed to vortex the container with the component for 30 seconds. Subsequently the sample is sonicated at 30-40 KHz 0.22?0.04 W/cm2 for 5 minutes and subsequently vortexed for a further 30 seconds. Culture tests are carried out by inoculating 0.5 ml of sonication liquid into blood culture bottles for aerobic and anaerobic microorganisms and incubated for 14 days at 36? 1?C. These samples are subcultured to blood agar, chocolate agar and Schaedler agar when the broth becomes cloudy or at the end of incubation. Real-time PCR is also performed using species-specific primers designed to assess bioburden. These techniques aim at the detection of a predetermined genus or species based on the clinical and epidemiological context.

Claims (6)

PATENT FOR INDUSTRIAL INVENTION TITLE: USE OF DYES IN THE SELECTIVE IDENTIFICATION OF INFECTED SITES IN BIOFILM-RELATED INFECTIONS 1 Formulations containing dyes and mixtures of dyes, such as but not limited to, fuchsin, eosin, methylene blue, toluidine blue O, in the form of spray, gel, emulsion, suspension, ointment, for clinical applications such as: i) osteomyelitis to guide surgical treatment; ii) biofilm-related infections of joint prostheses and fixation devices: for diagnostic and therapeutic purposes; iii) management of infected skin ulcers: for therapeutic purposes and/or guidance for debridement; iv) management of contaminated and/or infected wounds: for diagnostic and therapeutic purposes. 2 Formulations containing metal complexes of Ag, Cu, Au, Pd, Pt containing dyes such as, but not limited to, fuchsin, eosin, methylene blue, toluidine blue O and metal nanoparticles functionalized with these dyes, in the form of sprays, gels , emulsion, suspension, ointment, for clinical applications such as: i) osteomyelitis to guide surgical treatment; ii) biofilm-related infections of joint prostheses and fixation devices: for diagnostic and therapeutic purposes; iii) management of infected skin ulcers: for therapeutic purposes and/or guidance for debridement; iv) management of contaminated and/or infected wounds: for diagnostic and therapeutic purposes. 3 Formulations containing colorants and colorant mixtures, such as, for example and not only, fuchsin, eosin, methylene blue, toluidine blue O, in the form of spray, gel, emulsion, suspension, solution, for applications such as cleaning of surfaces and/or equipment and/or objects in general for verify its effective sanitization. 4 Formulations containing metal complexes of Ag, Cu, Au, Pd, Pt containing dyes such as, but not limited to, fuchsin, eosin, methylene blue, toluidine blue O and metal nanoparticles functionalized with these dyes, in the form of sprays, gels , emulsion, suspension, solution, for applications such as cleaning surfaces and/or equipment and/or objects in general to verify their effective sanitization. 5 Creation and use of a device dispensing the formulations referred to in claims 1, 2, 3, 4 capable of detecting in a simple and evident way the presence and type of bacteria and the concentration through a change in color visible to the eye, without additional tools. 6 Creation and use of a kit including the dispensing device referred to in claim 5 and a portable UV-VIS spectrophotometer, capable of detecting the type of bacteria and the concentration in a simple and evident way.