Antimicrobial susceptibility

Abstract

The present invention relates to methods and kits for the detection and determination of susceptibility of microorganisms to therapeutic agents. The present invention relates to the rapid determination of bacterial susceptibility to antibiotics. The present invention also extends to methods of sample preparation and microbial extraction from biological samples, as well as methods of identifying microorganisms, and kits and panels specifically designed to determine the susceptibility of extracted microorganisms to antimicrobial agents. The present invention is particularly useful in clinical diagnostics and veterinary medicine.

Description

[Technical field]

[0001] The present invention relates to methods and kits for the detection and determination of antimicrobial susceptibility and susceptibility of microorganisms to therapeutic agents, and in particular, but not exclusively, to the rapid determination of bacterial susceptibility to antibiotics. The invention also extends to methods of sample preparation and microbial extraction from biological samples, as well as methods of identifying microorganisms, and kits and panels specifically designed to determine the susceptibility of extracted microorganisms to antimicrobial agents. The invention is particularly useful in clinical diagnostics and veterinary medicine. [Background technology]

[0002] Antimicrobial resistance poses a serious global threat of increasing concern to human, animal, and environmental health. One of the main causes of antimicrobial resistance includes the overuse of antibiotics in animals and humans, and the release of unmetabolized antibiotics or their residues into the environment through compost or feces.

[0003] The antimicrobial susceptibility profile of a microorganism is usually determined by evaluating its growth ability in the presence of different antimicrobial agents, which usually takes about 2 days from the collection of a biological sample to the determination of the susceptibility profile. Epidemiologically based therapy through the application of broad-spectrum antimicrobial agents is applied in most hospitals around the world due to the long time required for these procedures. This then induces antimicrobial resistance leading to severe individual and public health threats.

[0004] Therefore, there is an urgent need for rapid antimicrobial susceptibility testing (AST) that can provide relevant and useful information in a timely manner that can enable optimal implementation of antimicrobial therapy based on scientific reports. To meet this increasing need, several approaches have been developed over the years, including molecular-based assays. Although molecular assays may have the advantage of directly screening polymicrobial samples for selected antimicrobial resistance mechanisms, they do not provide information on bacterial susceptibility to certain antimicrobial agents and therefore have a very limited role in patient treatment.

[0005] Moreover, molecular assays can only assess mechanisms of resistance, and evidence of discrepancies between genotypic and phenotypic test outcomes has been reported. Antimicrobial resistance is too complex to rely on such methods for a truly comprehensive understanding of the problem. Summary of the Invention [Means for solving the problem]

[0006] Therefore, to overcome the limitations of existing AST, the inventors have developed a new highly innovative method, called "FASTinov Assay", together with a corresponding platform, which allows to provide AST reports to clinicians directly from positive biological samples at the time of recording, thus resulting in the implementation of targeted antimicrobial therapy instead of the use of broad-spectrum antimicrobial agents [6, 7, 8]. The invention also allows the detection of multi-drug resistant microorganisms [5], which in turn allows the implementation of more aggressive therapy in a minimal amount of time, thereby avoiding their spread throughout the ward, hospital or beyond. Thus, the invention described herein has the potential to revolutionize the treatment of infected patients with significant benefits for both patients and society.

[0007] The present invention provides a major improvement and development of our previous susceptibility testing method and panel described in WO 2012 / 164547 A1 [1]. Our new method involves an innovative sample preparation and extraction method aimed at separating microorganisms from human or animal cells and debris from positive biological samples. The isolated microorganisms are then stained with an optimized fluorescent probe after being incubated with an antimicrobial drug for a very short period of time. The damage produced by the drug to the cells is then evaluated through a multiparameter analysis using a flow cytometer. As the test is growth independent, results are obtained at the time of recording.

[0008] Flow cytometry has proven to be a powerful tool in various research fields, such as hematology and cytopathology, and its increasing use in microbiology has significant potential. Indeed, cellular microbial populations can be differentiated in terms of susceptible versus resistant phenotypes in very short time frames using flow cytometry. Indeed, antimicrobial effects can be rapidly detected and quantified using precise fluorescent dyes at determined wavelengths and software that allows the analysis of large amounts of biological data, including cell size and complexity.

[0009] Thus, in a first aspect of the invention there is provided a method for determining the susceptibility phenotype of a microorganism present in a biological sample to at least one therapeutic agent, comprising the steps of: (i) A biological sample containing a microorganism, (a) one or more first test reservoirs; (b) one or more second positive control reservoirs, and (c) one or more third negative control reservoirs containing non-viable microorganisms. introducing into each of (ii) contacting the biological sample in one or more first test reservoirs with at least one therapeutic agent; (iii) contacting the biological sample in the one or more first, second and third reservoirs with at least one fluorescent marker; and (iv) performing a fluorescence analysis to obtain one or more fluorescence parameters for the biological sample in each of the reservoirs. Including, A susceptibility phenotype of the microorganism to at least one therapeutic agent is obtained by comparing one or more fluorescence parameters between the reservoirs. A method is provided.

[0010] Advantageously, the inventors have discovered that the use of a third negative control reservoir used in addition to a second positive control reservoir in the method significantly increases the sensitivity of the assay, resulting in a valid and accurate susceptibility phenotype readout, allowing optimal therapeutic treatment for the patient. This is because the use of both a positive control and a negative control in addition to the test sample ensures that only viable microorganisms are considered in the susceptibility phenotype assessment assay. Non-viable (i.e. dead) microorganisms cannot respond to therapeutic agents, and therefore, if they were included in the susceptibility phenotype assessment, such non-viable microorganisms may be erroneously seen as resistant to all therapeutic agents, which corresponds to a false negative or major error, resulting in the implementation of an incorrect subsequent therapy.

[0011] Preferably, therefore, the one or more third negative control reservoirs contain non-viable or dead microorganisms and the one or more second positive control reservoirs contain viable or live microorganisms. Preferably, the one or more first test reservoirs contain viable or live microorganisms.

[0012] Preferably, the one or more first test reservoirs contain at least 60%, 70%, or 80% viable cells. More preferably, the one or more first test reservoirs contain at least 85%, 90%, or 95% viable cells. More preferably, the one or more first test reservoirs contain at least 96%, 97%, 98%, 99%, or 100% viable cells. Preferably, the one or more first test reservoirs contain at least 10% viable cells. 5CFU / ml of viable cells or at least 10 6 CFU / ml of viable cells or at least 10 7 Contains viable cells CFU / ml.

[0013] Preferably, the one or more second positive control reservoirs contain at least 60%, 70%, or 80% viable cells. More preferably, the one or more first second positive control reservoirs contain at least 85%, 90%, or 95% viable cells. More preferably, the one or more second positive control reservoirs contain at least 96%, 97%, 98%, 99%, or 100% viable cells. Preferably, the one or more second test reservoirs contain at least 10% viable cells. 5 CFU / ml of viable cells or at least 10 6 CFU / ml of viable cells or at least 10 7 Contains viable cells CFU / ml.

[0014] Preferably, the one or more third negative control reservoirs contain at least 60%, 70%, or 80% non-viable cells. More preferably, the one or more third negative control reservoirs contain at least 85%, 90%, or 95% non-viable cells. More preferably, the one or more third negative control reservoirs contain at least 96%, 97%, 98%, 99%, or 100% non-viable cells.

[0015] The cells in the one or more third negative controls may be rendered non-viable by exposure to a cell killing agent. Preferably, the one or more third negative control reservoirs contain a cell killing agent. Preferably, the cell killing agent is selected from the group consisting of ethanol, 2-phenoxyethanol, citric acid, and benzydamine hydrochloride. Most preferably, the cell killing agent is benzydamine hydrochloride.

[0016] The inventors have previously shown that benzydamine hydrochloride rapidly kills bacteria

[14] and fungi

[13] by cell membrane damage. The purpose of this control is to ensure that the fluorescent probe is active and has the greatest effect when cells are killed by benzydamine hydrochloride. Thus, in a preferred embodiment, cells in one or more third negative control reservoirs are rendered non-viable by exposure to benzydamine hydrochloride.

[0017] If at least one fluorescent marker is not present or does not function well, it is difficult, if not impossible, to evaluate the effect of the therapeutic agent on the microorganism. Therefore, the use of a negative control reservoir enhances the accuracy of the method. Furthermore, the inventors have surprisingly discovered that the use of both a positive control and a negative control in the method of the present invention allows the identification of different levels of susceptibility phenotype to at least one therapeutic agent, i.e., high susceptibility, intermediate susceptibility or resistance to at least one therapeutic agent.

[0018] Thus, in one embodiment, the susceptible phenotype may be hypersusceptible, intermediate susceptible or resistant.

[0019] Fluorescence analysis may be performed by flow cytometry. Accurate measurement of the fluorescence signal by flow cytometry may be hindered by background fluorescence caused by the autofluorescence of the microorganism itself. To overcome this problem, the inventors have advantageously discovered that the use of a fourth autofluorescence control reservoir, which contains microorganisms that have not been treated with a therapeutic agent and that have not been stained with a fluorescent marker, allows for the evaluation of the autofluorescence of the microorganism itself. When taken into account during the analysis of the fluorescence parameters used to determine the susceptibility profile of the microorganism, the elimination of the autofluorescence background significantly increases the accuracy of the test.

[0020] Thus, in one embodiment, the method further comprises introducing the biological sample into one or more fourth autofluorescence control reservoirs, where no therapeutic agent or fluorescent marker is added to the reservoirs, in other words, the one or more fourth autofluorescence control reservoirs do not contain a therapeutic agent or a fluorescent marker.

[0021] Preferably, the one or more fourth autofluorescence control reservoirs contain viable or living microorganisms. Preferably, the one or more fourth autofluorescence control reservoirs contain at least 60%, 70%, or 80% viable cells. More preferably, the one or more fourth autofluorescence control reservoirs contain at least 85%, 90%, or 95% viable cells. More preferably, the one or more fourth autofluorescence control reservoirs contain at least 96%, 97%, 98%, 99%, or 100% viable cells. Preferably, the one or more fourth autofluorescence control reservoirs contain at least 10% viable cells. 5 CFU / ml of viable cells or at least 10 6 CFU / ml of viable cells or at least 10 7 Contains viable cells CFU / ml.

[0022] The inventors have surprisingly discovered that the sensitivity of the method is increased if the microorganisms are isolated or separated from any host cells present in the biological sample (i.e. host cells originating from the subject / patient from whom the sample is taken) prior to introduction into the test and control reservoirs. Without the use of a highly pure and concentrated sample of microbial cells (without the presence of any host cells from the test subject), accurate flow cytometry reading and analysis is difficult, as the host cells themselves can produce non-specific fluorescence, thereby interfering with the analysis of the fluorescence produced by the microorganisms.

[0023] Thus, in a preferred embodiment, the method further comprises a sample preparation step. Preferably, the sample preparation step comprises extracting and purifying the microorganisms from host cells and / or debris also present in the biological sample prior to introduction into one or more of the first, second, third and / or optionally fourth reservoirs.

[0024] The inventors have successfully demonstrated for the first time that a density gradient solution (e.g., histopaque® available from Sigma) capable of separating microorganisms from host cells and debris through a centrifugation gradient is much more effective than the standard physical separations used in the prior art. This novel method works with all types of biological samples without affecting the physical and physiological functions of the microorganisms, and therefore the fluorescence analysis is not affected.

[0025] Other methods for the direct identification of microorganisms from biological samples have been described in the art. However, these methods are limited to the use of hemolytic agents, and the use of density gradient solutions, such as histopaque®, has not been previously described in clinical microbiology methodology for the purpose of extracting and purifying microorganisms (e.g., bacteria) from biological samples. The novel method developed by the inventors allows the purified microorganisms to accumulate at the bottom of a tube or vial, while the cell debris and host cells, combined with histopaque®, accumulate at the top of the tube.

[0026] Moreover, the novel method developed by the inventors allows the same sample to be used for MALDI-TOF identification of bacteria causing infections. Generally, the prepared sample must undergo additional processing methods before processing for MALDI-TOF bacterial identification. However, when using the inventors' novel sample preparation method, the sample is automatically prepared for MALDI-TOF identification without requiring any further processing, thereby saving time, which is essential in the context of a clinical microbiology laboratory where timely diagnosis has a tremendous impact on the patient's prognosis.

[0027] Thus, the sample preparation step preferably includes purifying microorganisms from the biological sample before the biological sample is introduced into each of the first, second, third and / or optionally fourth reservoirs. (i) obtaining a biological sample containing a microorganism; and (ii) contacting the biological sample with a density gradient solution, thereby purifying the microorganisms; may include:

[0028] Preferably, the density gradient solution is histopaque®. It is preferably used pure, without any dilution. Histopaque® is a density gradient cell separation medium that includes Ficoll and sodium diatrizoate. Most preferably, the biological sample and the density gradient solution are in a ratio of about 1:1. The sample preparation step may include extracting microorganisms from the biological sample.

[0029] After contacting the biological sample with the density gradient solution, the sample preparation step then preferably includes centrifuging the sample, preferably for at least 1 minute, preferably at about 13,000 rpm. The sample preparation step includes resuspending the resulting pellet (containing the purified microorganisms) in a medium. The medium may preferably be a cation-adjusted broth, optionally about 1 ml of sterile and filtered Mueller Hinton II cation-adjusted broth.

[0030] In some embodiments, the sample preparation step may comprise contacting the biological sample with a lysing agent prior to contacting the biological sample with the density gradient solution. Preferably, the lysing agent is configured to lyse any contaminating cells in the sample, such as contaminating host cells emanating from the host from which the sample is taken, or debris present in an environmental sample.

[0031] The inventors have found that the use of a hemolytic agent is particularly advantageous in embodiments in which the biological sample may comprise blood, preferably a blood culture. Protocol B disclosed in Figure 1 illustrates one preferred embodiment of the sample preparation step of the method when the biological sample is a blood sample.

[0032] In one embodiment, the lysing agent may be Triton® X-100 (commonly referred to as Triton X-100). Triton® X-100 is a common non-ionic detergent and emulsifier often used in biochemical applications to solubilize proteins. It is considered to be a relatively mild and non-denaturing detergent. It is utilized to lyse cells to extract proteins and organelles. It can also permeabilize living cell membranes for transfection or can be used for DNA extraction.

[0033] Preferably, Triton X-100 is used at a concentration of 0.1% to 3% (v / v), or 0.2% to 2.5% (v / v), or 0.3% to 2% (v / v), or 0.4 to 1% (v / v), preferably about 0.5% (v / v). Preferably, the biological sample is contacted with the hemolytic agent (preferably Triton X-100) for at least 1 minute, 2 minutes or 5 minutes. Preferably, the biological sample is contacted with the hemolytic agent (preferably Triton X-100) for less than 2 hours, less than 1 hour, less than 30 minutes or less than 15 minutes. This step may be carried out at room temperature. Thus, preferably, the sample preparation step comprises a step of contacting the biological sample with the hemolytic agent at room temperature for 5 minutes.

[0034] In another preferred embodiment, the lysing agent is Tergitol™ 15-S-9 (commonly referred to as Tergitol). Tergitol is a secondary alcohol ethoxylate and a linear non-ionic surfactant.

[0035] Preferably, Tergitol is used at a concentration of 2.5-25% (v / v), or 3%-22% (v / v), or 4%-20% (v / v), or 5-18% (v / v). More preferably, Tergitol is used at a concentration of 6% and 16% (v / v), or 6%-16% (v / v), or 7% and 14% (v / v), or 7%-14% (v / v). Most preferably, Tergitol is used at a concentration of 8-12% (v / v), or 9-11% (v / v), preferably about 10% (v / v). Preferably, the biological sample is contacted with Tergitol and mixed (preferably by vortexing).

[0036] Advantageously, Tergitol has a better safety profile since it is not listed on the REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) list of hazardous substances. Moreover, Tergitol requires only very short incubation times, thus ensuring a time-efficient diagnosis. Additionally, Tergitol functions advantageously in MALDI-Tof identification. Tergitol also ensures that samples of a higher degree of purity are analysed. Thus, the use of Tergitol is compatible with flow cytometry (FC) analysis, which requires extremely pure cell suspensions.

[0037] After contacting the biological sample with the hemolysing agent and before contacting the biological sample with the density gradient solution, the sample preparation step preferably includes centrifuging the sample, preferably for at least 1 minute, preferably at about 13,000 rpm. Preferably, the sample preparation step then includes resuspending the resulting pellet (containing the microorganisms). Preferably, the pellet is resuspended in saline solution, optionally about 0.05 ml to 2 ml, 0.01 ml to 1 ml, or 0.25 ml to 0.75 ml of sterile and filtered saline solution. Most preferably, the pellet is resuspended in 0.5 ml of sterile and filtered saline solution.

[0038] The lysing agent used in the extraction method makes it possible to lyse the host cells, in particular red blood cells, and potential debris that may be present in the biological sample, while preserving the microbial cells. The sample preparation protocol described herein makes it possible for the purified microorganisms to accumulate at the bottom of the tube, while the cellular debris and the host cells, combined with the density gradient solution, accumulate at the top of the tube.

[0039] The sample preparation step may further include identifying the microorganisms prior to introducing the biological sample containing the microorganisms into each of the first, second, third and / or optionally fourth reservoirs. The identification of the microorganisms may be performed using genetic methods, microarrays, physical methods and / or mass spectrometry methods. The genetic methods may be quantitative polymerase chain reaction (PCR), immuno-PCR, or a combination thereof. The microarrays may be DNA microarrays, protein microarrays, antibody microarrays, or a combination thereof. The physical methods may be infrared and Raman spectroscopy or laser-induced breakdown spectroscopy (LIBS). The mass spectrometry methods may be performed through an ICP mass spectrometer, a DART mass spectrometer, or a MALDI-TOF Brucker.

[0040] Preferably, the identification of the microorganisms is carried out using mass spectrometry methods, most preferably using MALDI-TOF, for example from manufacturers such as Brucker.

[0041] Advantageously, as shown in Figures 6A and 6B, the inventors have discovered that using samples extracted and purified using the above sample preparation steps for subsequent identification of microorganisms rapidly (i.e., within just a few hours) produces outstanding results and superior identification accuracy comparable to that obtained from standard microbial colonies, without the extra step of generating colonies from overnight cultures.

[0042] Thus, in one embodiment, identification of the microorganism is performed using a sample purified using the sample preparation steps described above. Preferably, the purified sample is substantially dried before being exposed to a mass spectrometer.

[0043] Thus, the sample preparation step may further comprise an additional centrifugation cycle to dry the pellet. The additional centrifugation cycle may be carried out for 5 seconds to 5 minutes, 10 seconds to 5 minutes, 30 seconds to 5 minutes, 30 seconds to 4 minutes, 30 seconds to 3 minutes, 30 seconds to 2 minutes, 30 seconds to 1.5 minutes, or 30 seconds to 1 minute. Preferably, the additional centrifugation cycle may be carried out for at least 1 minute.

[0044] The additional centrifugation step may be carried out at 5000 rpm to 20000 rpm, 10000 rpm to 18000 rpm, 12000 rpm to 16000 rpm, or 13000 rpm to 15000 rpm.

[0045] The sample preparation step may further include drying the pellet resulting from the additional centrifugation cycle. The resulting pellet may be dried at 0° C. to 40° C., or 30° C. to 40° C., or 35° C. to 40° C. The resulting pellet may be dried at 4° C., room temperature, or 37° C.

[0046] Other sample purification methods are also contemplated to be within the scope of the present invention for microbial identification, including, for example, methods recommended by mass spectrometer manufacturers.

[0047] The inventors have successfully demonstrated that the novel sample preparation process described above advantageously provides a very rapid, one-step method for obtaining highly purified samples for efficient identification of microorganisms and then subsequent testing of their susceptibility to antimicrobial agents in a time-efficient manner.

[0048] The biological sample may be of human, animal or environmental origin. In embodiments where the biological sample is of human or animal origin, the sample is preferably a biological sample taken from a test subject. The method for determining the susceptibility phenotype of a microorganism in a sample is therefore preferably carried out in vitro or ex vivo. The sample may comprise tissue, blood, plasma, serum, spinal fluid, urine, bronchial secretions, cerebrospinal fluid, sweat, saliva, sputum, tears, breast aspirate, prostatic fluid, semen, vaginal fluid, feces, cervical scrapings, amniotic fluid, intraocular fluid, mucus, breath moisture, animal tissue, cell lysate, tumor tissue, hair, skin, buccal scrapings, nails, bone marrow, cartilage, prions, bone powder, ear wax, or combinations thereof. The sample may be a biopsy. Preferably, the sample is blood. Preferably, the sample is saliva.

[0049] In a preferred embodiment, the biological sample of human origin is a urine sample or a blood sample. A blood sample is preferred. Most preferably, the sample is a blood culture.

[0050] In embodiments where the biological sample is of environmental origin, the sample is preferably selected from the group consisting of soil; water; and plant residues.

[0051] The biological sample may or may not be cultured before being subjected to the analysis of the method of the present invention. Advantageously, the ability to process the biological sample when not cultured significantly reduces the experimental time and avoids unnecessary sample incubation steps. Thus, preferably, the biological sample is not cultured.

[0052] The biological material may be an aerobic or anaerobic sample. The sample may thus comprise an aerobic blood culture or an anaerobic blood culture.

[0053] Prior to fluorescence analysis, the one or more first, second, third and / or optionally fourth reservoirs may be incubated at 0° C.-40° C., 20° C.-40° C., 22° C.-40° C., 24° C.-40° C., 26° C.-40° C., 28° C.-40° C., 30° C.-40° C., 32° C.-40° C., 34° C.-40° C., 36° C.-40° C., or 38° C.-40° C. The one or more first, second, third and / or fourth reservoirs may be incubated at 0° C.-40° C., or 30° C.-40° C., or 35° C.-40° C., or at about 0° C., 4° C., room temperature, or 37° C. Preferably, the one or more first, second, third and / or fourth reservoirs are incubated at 37° C.

[0054] Prior to fluorescence analysis, one or more of the first, second, third and / or optionally fourth reservoirs may be incubated with or without shaking. Preferably, the biological sample is incubated with shaking.

[0055] Prior to fluorescence analysis, the one or more first, second, third, and / or optionally fourth reservoirs may be incubated for up to 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, or 30 minutes. Preferably, the biological sample is incubated for about 1 hour.

[0056] After this incubation step, the one or more of the first, second, third and / or fourth reservoirs are then preferably subjected to a fluorescence analysis step.

[0057] The microorganism may be a bacterium, a virus, a fungus or a protozoan.

[0058] In a preferred embodiment, the microorganism is a bacterium. The bacterium may be a gram-negative bacterium, a gram-positive bacterium, or a bacterium that exhibits characteristics of both gram-negative and gram-positive bacteria. The bacterium may be aerobic or anaerobic. In a preferred embodiment, the microorganism is an aerobic bacterium.

[0059] Preferably, the gram-negative bacterium is Escherichia coli ATCC 25922; Escherichia coli ATCC 8739; Escherichia coli ATCC 35218; Escherichia coli BAA 2425; Klebsiella pneumoniae ATCC 13443; Klebsiella pneumoniae BAA 1705; Klebsiella pneumoniae ATCC 700603; Klebsiella pneumoniae BAA1706; Enterobacter aerogenes ATCC 13048; Serratia marcescens ATCC 14756; Providencia rettgeri BAA 2525; Pseudomonas aeruginosa 27853; Pseudomonas aeruginosa BAA 2108; and Acinetobacter baumannii BAA1709.

[0060] Preferably, the gram-positive bacterium is a gram-positive cocci in the grape-like cluster selected from the group consisting of Staphylococcus aureus 29213; Staphylococcus aureus 43300; Staphylococcus aureus 700698; and Staphylococcus epidermidis 35984.

[0061] More preferably, the gram-positive bacterium may also be a gram-positive cocci in a chain selected from the group consisting of Enterococcus faecalis 29212; Enterococcus faecalis 51299; Enterococcus faecium 700221; Enterococcus casseliflavus 700668; and Enterococcus gallinarum 49608.

[0062] Bacteria also include Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, Bordetella pertussis, Burkholderia spp. (e.g., Burkholderia mallei, Burkholderia pseudomallei, and Burkholderia cepacia), Haemophilus influenzae, Clostridium tetani (tetanus), Clostridium perfringens, and others. perfringens, Clostridium botulinum, Corynebacterium diphtheriae (diphtheria), Legionella pneumophila, Coxiella burnetii, Brucella spp. (e.g., B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis, B. suis, and B. pinnipediae), Francisella spp. (e.g., F. novicida, F. filomilagia, F. philomiragia) and F. tularensis (F.tularensis), Streptococcus agalactiae, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum (syphilis), Haemophilus ducreyi, Helicobacter pylori, Staphylococcus saprophyticus, Yersinia enterocolitica, Bacillus anthracis (anthrax), Yersinia pestis (plague), Mycobacterium tuberculosis tuberculosis, Rickettsia, Listeria, Chlamydia pneumoniae, Vibrio cholerae, Salmonella typhi (typhoid), Borrelia burgdorferi, Porphyromonas, Streptococcus pneumoniae, Mycobacterium tuberculosis and Haemophilus influenzae.

[0063] In another embodiment, the microorganism may be a virus, which may be selected from the group consisting of orthomyxoviruses, Paramyxoviridae viruses, metapneumoviruses and morbilliviruses, pneumoviruses, paramyxoviruses, Poxviridae viruses, metapneumoviruses, morbilliviruses, picornaviruses, enteroviruses, bunyaviruses, phleboviruses, nairoviruses, heparnaviruses, togaviruses, alphaviruses, arteriviruses, flaviviruses, pestiviruses, hepadnaviruses, rhabdoviruses, Caliciviridae viruses, coronaviruses, retroviruses, reoviruses, parvoviruses, hepatitis delta virus (HDV), hepatitis E virus (HEV), human herpesviruses and papovaviruses.

[0064] The orthomyxovirus may be influenza A, B and C. The paramyxoviridae virus may be pneumovirus (RSV), paramyxovirus (PIV). The metapneumovirus may be morbillivirus (e.g. measles). The pneumovirus may be respiratory syncytial virus (RSV), bovine respiratory syncytial virus, pneumonia virus of mice, or turkey rhinotracheitis virus. The paramyxovirus may be parainfluenza virus types 1-4 (PIV), mumps, Sendai virus, Simian virus 5, bovine parainfluenza virus, Nipah virus, Henipa virus, or Newcastle disease virus. The poxvirus may be smallpox virus (Variola vera), such as variola major virus and variola minor virus. The metapneumovirus may be human metapneumovirus (hMPV) or avian metapneumovirus (aMPV). The morbillivirus may be measles virus. The picornavirus may be an enterovirus, a rhinovirus, a heparnavirus, a parechovirus, a cardiovirus, or an aphthovirus. The enterovirus may be a poliovirus type 1, 2, or 3, a coxsackie A virus type 1-22 and 24, a coxsackie B virus type 1-6, an echovirus (ECHO virus) type 1-9, 11-27, and 29-34, or an enterovirus type 68-71. The bunyavirus may be a California encephalitis virus. The phlebovirus may be a Rift Valley fever virus. The nairovirus may be a Crimean-Congo hemorrhagic fever virus. The heparnavirus may be a hepatitis A virus (HAV). The togavirus may be a rubivirus. The flavivirus may be tick-borne encephalitis (TBE) virus, dengue (types 1, 2, 3 or 4) virus, yellow fever virus, Japanese encephalitis virus, Kyasanur Forest virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus or Powassan encephalitis virus. The pestivirus may be bovine viral diarrhea (BVDV), swine fever (CSFV) or border disease virus (BDV). The hepadnavirus may be hepatitis B virus or hepatitis C virus.The rhabdovirus may be a lyssavirus (rabies virus) or a vesiculovirus (VSV). The caliciviridae virus may be a Norwalk virus or a Norwalk-like virus, such as Hawaii virus and Snow Mountain virus. The coronavirus may be SARS CoV-1, SARS-CoV-2, MERS, human respiratory coronavirus, avian infectious bronchitis (IBV), mouse hepatitis virus (MHV), or transmissible gastroenteritis virus of porcine (TGEV). The retrovirus may be an oncovirus, lentivirus, or spumavirus. The reovirus may be an orthoreovirus, rotavirus, orbivirus, or coltivirus. The parvovirus may be parvovirus B19. The human herpesvirus may be herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), or human herpesvirus 8 (HHV8). The papovavirus may be a papillomavirus, polyomavirus, adenovirus, or arenavirus. Preferably, the virus is selected from the group consisting of SARS CoV, SARS CoV2, MERS, or influenza.

[0065] However, in another embodiment, the microorganism may be a fungus, such as Epidermophyton floccosum, Microsporum audouinii, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton nigricans ... from dermatophytes, including Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and / or Trichophyton faviforme; or Aspergillus fumigatus. fumigatus, Aspergillus flavus, Aspergillus nigerniger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowii, Aspergillus clavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea stellatoidea, Candida krusei, Candida paracasei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondii, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum capsulatum, Klebsiella pneumoniae, Microsporidia, Encephalitozoon fungi, Septata intestinalis and Enterocytozoon bienui.bieneusi; Brachiola, Microsporidium, Nosema, Pleistophora, Trachipleistophora, Vittaforma Paracoccidioides brasiliensis, Pneumocystis carinii, Pythium insidiosum, Pityrosporum ovale, Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces pombe pombe, Scedosporium apiospermum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffeimarneffei, Malassezia fungi, Fonsecaea fungi, Wangiella fungi, Sporothrix fungi, Basidiobolus fungi, Conidiobolus fungi, Rhizopus fungi, Mucor fungi, Absidia fungi, Mortierella fungi, Cunninghamella fungi, Saksenaea fungi, Arte The fungus may be selected from the group consisting of the fungi of the genus Alternaria, Curvularia, Helminthosporium, Fusarium, Aspergillus, Penicillium, Monilinia, Rhizoctonia, Paecilomyces, Pithomyces, and Cladosporium. Preferably, the fungus is selected from the group consisting of Aspergillus, Cryptococcus, or Pneumocystis.

[0066] In yet another embodiment, the microorganism may be a protozoan, which may be selected from the group consisting of Entamoeba histolytica, Giardia lamblia, Cryptosporidium parvum, Cyclospora cayetanensis, and Toxoplasma.

[0067] In one embodiment, the therapeutic agent is an antibiotic, an antiviral, an antifungal, or an antiprotozoan agent. Preferably, the therapeutic agent is an antibiotic.

[0068] Preferably, the selection of the antimicrobial agent may be based on bacterial Gram staining in accordance with the protocols of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical & Laboratory Standard Institute (CLSI). Hence, the therapeutic agent is preferably an antimicrobial agent.

[0069] Thus, for gram-negative bacteria, the antimicrobial agent may be selected from the group consisting of amikacin; gentamicin, ciprofloxacin, imipenem, meropenem, ertapenem, ceftazidime-avibactam, piperacillin-tazobactam, cefepime, ceftazidime, cefotaxime, ceftazidime-clavulanate, cefotaxime-clavulanate, ceftolozane-tazobactam, amoxicillin-clavulanate, ampicillin, fosfomycin, nitrofurantoin, and colistin.

[0070] For gram-positive bacteria, the antimicrobial agent may be selected from the group consisting of penicillin, ampicillin, cefoxitin, oxacillin, imipenem, vancomycin, linezolid, gentamicin, high level gentamicin, levofloxacin, and daptomycin.

[0071] Preferably, the antifungal agent may be selected from the group consisting of caspofungin, micafungin, anidulafungin, posaconazole, voriconazole, flucytosine, amphotericin B, itraconazole, posaconazole and fluconazole.

[0072] Preferably, the antiviral agent may be selected from the group consisting of protease inhibitors, such as ritonavir, atazanavir or darunavir; inhibitors of viral DNA polymerase, such as acyclovir, tenofovir, valganciclovir or valacyclovir; and inhibitors of integrase, such as raltegravir.

[0073] Preferably, the antiprotozoal agent may be selected from the group consisting of metronidazole, atovaquone, benznidazole, dehydroemetine, eflornithine, emetine, fenbendazole, iodoquinol, melarsoprol, nifurtimox, pentamidine, quinacrine, sodium stibogluconate, suramin, and tinidazole.

[0074] The fluorescence analysis used in the methods of the invention may be a flow cytometric analysis or a laser scanning analysis. The flow cytometer may be equipped with a plate reader for automated analysis of each test reservoir. The flow cytometer may also be equipped with a blue laser and / or have three fluorescence channels.

[0075] In a preferred embodiment, the one or more flow cytometry parameters include forward scatter and / or side scatter and / or fluorescence parameters. The fluorescence scatter signal may be intensity, a spectral profile and / or a cell count.

[0076] The fluorescent marker may be selected from the group consisting of nucleic acid stains; metabolic stains; membrane potential stains; probes for cell organelles; fluorescent tracers of cell morphology and fluid flow; probes for cell viability, proliferation and function; and / or probes for reactive oxygen species.

[0077] Preferably, the fluorescent marker may be selected from the group consisting of acridine dyes, cyanine dyes, fluorone dyes, oxazine dyes, phenanthridine dyes, or rhodamine dyes. Most preferably, the fluorescent marker may be selected from the group consisting of CTC (5-cyano-2,3-ditolyltetrazolium chloride), calcein AM, dihydrorhodamine 123, DIBAC4(3), DioC 2(3), fluorescein diacetate, 5CFDA, AM, CFDA-SE, propidium iodide, SYTO 16 green fluorescence, and nucleic acid stains.

[0078] In a preferred embodiment, the fluorescent marker DiBAC4(3) is combined with the antibiotics listed in FIG. 3A to test the susceptibility of Gram-negative bacteria to these antibiotics.

[0079] Preferably, the fluorescent marker propidium iodide (PI) is combined with the antibiotic imipenem to test the susceptibility of Pseudomonas species to imipenem.

[0080] Most preferably, the fluorescent marker propidium iodide (PI) may be combined with these antibiotics to test the susceptibility of Gram-positive bacteria to the antibiotics listed in FIG. 4A.

[0081] The fluorescent marker DiOC2(3) (3,3'-diethyloxacarbocyanine iodide) may be combined with these antibiotics to test the susceptibility of Gram-positive bacteria to these antibiotics as described in Figure 4A.

[0082] Preferably, the fluorescent marker propidium iodide (PI) may be combined with colistin to test the susceptibility of Gram-positive bacteria to colistin.

[0083] In one embodiment, the method of the invention comprises: (i) one or more first test reservoirs containing a fluorescent marker / therapeutic agent combination; (ii) one or more second positive control reservoirs, and (iii) one or more third negative control reservoirs, both containing the same fluorescent marker, and optionally (iv) one or more fourth autofluorescent control reservoirs in which neither the fluorescent marker nor the therapeutic agent is present. may include at least one set of

[0084] In other words, preferably at least one combination of fluorescent marker / therapeutic agent (preferably fluorescent marker / antibiotic) is tested, in which the fluorescent markers in the set are the same.

[0085] In another embodiment, the method of the first aspect comprises: (i) one or more first test reservoirs containing a fluorescent marker / therapeutic agent combination; (ii) one or more second positive control reservoirs, and (iii) one or more third negative control reservoirs, both containing the same fluorescent marker, and optionally (iv) one or more fourth autofluorescent control reservoirs in which neither the fluorescent marker nor the therapeutic agent is present. Includes multiple sets of.

[0086] In other words, preferably at least 2, 3, 4 or more different combinations of fluorescent marker / therapeutic agent (preferably fluorescent marker / antibiotic) are tested. Preferably at least 5, 6, 7 or more different combinations of fluorescent marker / therapeutic agent (preferably fluorescent marker / antibiotic) are tested. Within each set, the fluorescent marker is the same. However, the fluorescent marker may be the same or different among the sets. The number and type of fluorescent marker / therapeutic agent combinations may depend on whether Gram-negative or Gram-positive bacteria are being tested.

[0087] The reservoir may be a container, a tube or a well. Preferably, the reservoir is a well. For example, the well may be part of a plate, such as a 96-well plate.

[0088] In a second aspect of the invention there is provided a kit for use in the method of the first aspect.

[0089] The kit of the invention may be a panel comprising one or more first, second, third and / or fourth reservoirs. Preferably, the panel may be a 96-well plate.

[0090] Advantageously, the inventors have carefully designed test panels optimized for the detection of various microorganisms. Each test panel contains a carefully selected combination of therapeutic agents / antibiotics and fluorescent markers, and each therapeutic agent / antibiotic and fluorescent marker / fluorescent dye combination is optimized to increase the accuracy of the readout. The present invention marks the first disclosure of pre-set combinations of drugs and fluorescent dyes in a test panel prior to incubating the microorganisms.

[0091] In one embodiment, the panel may be the FASTgramneg panel disclosed in Figure 3A. Preferably, the FASTgramneg panel includes wells as disclosed in Figure 3B. Thus, preferably, the panel is configured to analyze Gram-negative bacteria.

[0092] Preferably, in this panel for analyzing Gram-negative bacteria, the antibiotics ampicillin, amoxicillin-clavulanate, cefotaxime, ceftazidime, cefotaxime-clavulanate, ceftazidime-clavulanate, ceftolozane-tazobactam, cefepime, cefoxitin, ceftazidime-avibactam, piperacillin-tazobactam, ciprofloxacin, gentamicin, amikacin, nitrofurantoin, fosfomycin, imipenem and meropenem are preferably combined with DIBAC. Preferably, imipenem is combined with a PI (in a separate well).

[0093] In another embodiment, the panel may be the FASTgrampos panel disclosed in FIG. 4A. Preferably, the FASTgrampos panel comprises a well as disclosed in FIG. 4B. Preferably, the panel is thus configured for analyzing Gram-positive bacteria. Preferably, in this panel for analyzing Gram-positive bacteria, the antibiotics ampicillin, cefoxitin, oxacillin, penicillin, vancomycin and imipenem are preferably combined with a PI. Preferably, gentamicin, linezolid, daptomycin, levofloxacin are combined with a DIOC.

[0094] In yet another embodiment, the panel may be the FASTcolistin MIC panel disclosed in Figure 5A. Preferably, the FASTcolistin MIC panel comprises wells as disclosed in Figure 5B. Preferably, the panel is thus configured for analyzing Gram-negative bacteria. Preferably, in this panel for analyzing Gram-negative bacteria, colistin MIC is combined with a PI.

[0095] The inventors believe that the above-mentioned method for purifying microorganisms is novel. As mentioned above, the present disclosure constitutes the first evaluation of the use of density gradient solutions (e.g., histopaque®) in microbiological assays for purifying, identifying and / or diagnosing microorganisms. The inventors have demonstrated that this method is surprisingly much more effective than the standard physical separation used in the prior art, there is no loss of cells during centrifugation, and the suspension is much cleaner and therefore more pure.

[0096] Thus, in a third aspect of the present invention there is provided a method for purifying a microorganism from a biological sample comprising the steps of: (i) obtaining a biological sample containing a microorganism; and (ii) contacting the biological sample with a density gradient solution, thereby purifying the microorganisms; A method is provided, comprising:

[0097] In a fourth aspect there is therefore provided the use of a density gradient solution for purifying microorganisms from a biological sample.

[0098] The microorganism may be purified in the biological sample. The method may comprise the subsequent step of extracting the microorganism from the biological sample.

[0099] Preferably, the density gradient solution is histopaque (registered trademark-1077). Histopaque™ is a density gradient cell separation medium that includes Ficoll and sodium diatrizoate. Most preferably, the biological sample and the density gradient solution are in a 1:1 ratio.

[0100] After contacting the biological sample with the density gradient solution, the sample preparation step then preferably includes centrifuging the sample, preferably for at least 1 minute, preferably at about 13,000 rpm. The sample preparation step includes resuspending the resulting pellet (containing the purified microorganisms) in a medium. The medium may preferably be a cation-adjusted broth, optionally about 1 ml of sterile and filtered Mueller Hinton II cation-adjusted broth.

[0101] The method preferably includes a step of contacting the biological sample with a hemolytic agent before contacting the biological sample with the density gradient solution. Preferably, the hemolytic agent is configured to dissolve any contaminating cells in the sample, such as contaminating host cells emanating from the host from which the sample is taken, or debris present in an environmental sample. The use of a hemolytic agent is preferred in embodiments in which the sample comprises blood, preferably a blood culture. Protocol B disclosed in Figure 1 illustrates one preferred embodiment of the sample preparation step of the method when the biological sample is a blood sample.

[0102] In one embodiment, the lysing agent may be Triton® X-100 (commonly referred to as Triton X-100). Triton® X-100 is a common non-ionic detergent and emulsifier often used in biochemical applications to solubilize proteins. It is considered to be a relatively mild and non-denaturing detergent. It is utilized to lyse cells to extract proteins and organelles. It can also permeabilize living cell membranes for transfection or can be used for DNA extraction.

[0103] Preferably, Triton X-100 is used at a concentration of 0.1% to 3% (v / v), or 0.2% to 2.5% (v / v), or 0.3% to 2% (v / v), or 0.4 to 1% (v / v), preferably about 0.5% (v / v). Preferably, the biological sample is contacted with the hemolytic agent (preferably Triton X-100) for at least 1 minute, 2 minutes or 5 minutes. Preferably, the biological sample is contacted with the hemolytic agent (preferably Triton X-100) for less than 2 hours, less than 1 hour, less than 30 minutes or less than 15 minutes. This step may be carried out at room temperature. Therefore, preferably, the same preparation step includes a step of contacting the biological sample with the hemolytic agent at room temperature for 5 minutes.

[0104] After contacting the biological sample with the hemolysing agent and before contacting the biological sample with the density gradient solution, the sample preparation step preferably includes centrifuging the sample, preferably for at least 1 minute, preferably at about 13,000 rpm. Preferably, the sample preparation step then includes resuspending the resulting pellet (containing the microorganisms). Preferably, the pellet is resuspended in saline solution, optionally about 0.05 ml to 2 ml, 0.01 ml to 1 ml, or 0.25 ml to 0.75 ml of sterile and filtered saline solution. Most preferably, the pellet is resuspended in 0.5 ml of sterile and filtered saline solution.

[0105] The lysing agent used in the extraction method makes it possible to lyse the host cells, in particular red blood cells, and any potential debris that may be present in the biological sample, while preserving the microbial cells. The method described herein makes it possible for the purified microorganisms to accumulate at the bottom of the tube, while the cellular debris and the host cells, combined with the density gradient solution, accumulate at the top of the tube. Preferably, the method comprises a step of extracting the microorganisms from the sample.

[0106] The method may further comprise a step of identifying the microorganism prior to the step of introducing the biological sample containing the microorganism into each of the first, second, third and / or fourth reservoirs defined in the first aspect. The identification of the microorganism may be performed using genetic methods, microarrays, physical methods and / or mass spectrometry methods. The genetic methods may be quantitative polymerase chain reaction (PCR), immuno-PCR, or a combination thereof. The microarrays may be DNA microarrays, protein microarrays, antibody microarrays, or a combination thereof. The physical methods may be infrared and Raman spectroscopy or laser-induced breakdown spectroscopy (LIBS). The mass spectrometry methods may be performed through an ICP mass spectrometer, a DART mass spectrometer, or a MALDI-TOF Brucker.

[0107] Therefore, in a preferred embodiment, the extraction method of the first aspect comprises: (i) obtaining a biological sample containing a microorganism; (ii) contacting the sample with a density gradient solution; (iii) centrifuging the sample; and (iv) resuspending the resulting pellet Includes.

[0108] Preferably, the extraction method further comprises, between steps (i) and (ii), (a) contacting a biological sample with a hemolysing agent; (b) centrifuging the sample; and (c) optionally, resuspending the resulting pellet Further includes.

[0109] Therefore, in a further aspect, there is provided a method for determining the susceptibility phenotype of a microorganism present in a biological sample to at least one therapeutic agent, comprising the steps of: (i)(a) obtaining a biological sample containing a microorganism; (b) contacting the sample with a density gradient solution; and (c) optionally identifying the microorganism a sample preparation step comprising: (ii) a biological sample containing the microorganism, (d) one or more first test reservoirs; (e) one or more second positive control reservoirs, and (f) one or more third negative control reservoirs containing non-viable microorganisms; (g) one or more fourth autofluorescent control reservoirs. introducing into each of (iii) contacting the biological sample in the one or more first test reservoirs with at least one therapeutic agent; (iv) contacting the biological sample in the one or more first, second and third reservoirs with at least one fluorescent marker; and (v) performing a fluorescence analysis to obtain one or more fluorescence parameters for the biological sample in each of the reservoirs. Including, A susceptibility phenotype of the microorganism to at least one therapeutic agent is obtained by comparing one or more fluorescence parameters between the reservoirs. A method is provided.

[0110] Preferably, the density gradient solution is histopaque®.

[0111] Preferably, the extraction method of the first aspect further comprises, between steps (a) and (b): (i) contacting the biological sample with a hemolysing agent Further includes.

[0112] In one embodiment, the lysing agent may be incubated with the biological sample for less than or at least 10 minutes, less than or at least 9 minutes, less than or at least 8 minutes, less than or at least 7 minutes, less than or at least 6 minutes, less than or at least 5 minutes, less than or at least 4 minutes, less than or at least 3 minutes, less than or at least 2 minutes, or less than or at least 1 minute.

[0113] In another embodiment, the lysing agent may be incubated with the biological sample for less than 30 seconds, 20 seconds, 10 seconds, or 5 seconds. Preferably, the lysing agent may be incubated with the biological sample for less than 4 seconds, 3 seconds, 2 seconds, or 1 second.

[0114] Preferably, the hemolytic agent is Triton-X100.

[0115] In another preferred embodiment, the hemolytic agent is Tergitol.

[0116] Preferably, Tergitol is used at a concentration of 10%. Preferably, the biological sample is contacted with Tergitol and mixed (preferably by vortexing).

[0117] All of the features described in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above embodiments in any combination, except where at least some of the features and / or steps are mutually exclusive.

[0118] For a better understanding of the present invention, and to show how an embodiment thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: [Brief description of the drawings]

[0119] [Figure 1]FIG. 1 shows a schematic representation of two embodiments of a sample extraction protocol (Protocol A and Protocol B). [Diagram 2] FIG. 2 is a summary table of chemicals tested to kill cells contained in a negative control for the fluorescent probe used in one embodiment of the method of the present invention. [Figure 3A] FIG. 3A shows the layout of one embodiment of a FASTgramneg panel according to the present invention for testing for gram-negative bacteria. [Figure 3B] FIG. 3B shows a summary table of the composition of each well of the FASTgramneg panel shown in FIG. 3A. [Figure 4A] FIG. 4A discloses the layout of one embodiment of a FASTgrampos panel according to the present invention for testing Gram-positive bacteria. [Figure 4B] FIG. 4B shows a summary table of the composition of each well of the FASTgrampos panel shown in FIG. 4A. [Figure 5A] FIG. 5A discloses the layout of one embodiment of a FASTcolistin MIC panel according to the present invention for testing Gram-negative bacteria. [Figure 5B] FIG. 5B shows a summary table of the composition of each well of the FASTcolistin MIC panel shown in FIG. 5A. [Figure 6A] FIG. 6A shows the number of each species of Gram-negative bacteria correctly identified in MALDI-TOF from blood culture samples purified through the sample preparation process of the present invention compared to that obtained from colony forming units derived from overnight (24 hour) subcultures of the blood samples. [Figure 6B] FIG. 6B shows the number of Gram-positive bacteria of each species correctly identified in MALDI-TOF from blood culture samples purified through the sample preparation process of the present invention compared to that obtained from colony forming units derived from overnight (24 hour) subcultures of the blood samples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0120] The inventors have developed a novel method or assay for determining the susceptibility phenotype of microorganisms (e.g. bacteria, fungi, viruses or protozoa) from a biological sample to at least one therapeutic agent, e.g. an antibiotic. The method is referred to as the "FASTinov susceptibility assay". The inventors have also developed a kit specifically designed for the evaluation of antimicrobial susceptibility to several antibiotics by flow cytometry, called the "FASTinov panel". As part of the sample preparation process for use in the assay and kit, different methods of microbial extraction and purification were tested to optimize sample purity for subsequent analysis on the panel. In the process, the inventors have developed an optimized protocol that ensures optimal sample quality and purity.

[0121] To evaluate the robustness of their method, we tested two different blood samples spiked each time with one of the 30 most common bacterial isolates commonly found in clinical isolates (from the FASTinov collection). Depending on the isolate under analysis, optimized combinations of fluorescent probes and antibiotics were used.

[0122] The inventors have further designed an innovative panel layout that incorporates different optimized features of the present invention.

[0123] Materials and Methods (i) Sample preparation microorganisms Thirty bacteria belonging to the FASTinov collection and representing the most common bacterial isolates found in clinical settings were selected for this study. These isolates consisted of both ATCC and clinical strains. Among them, 10 isolates were Gram-negative bacilli, 10 were Gram-positive cocci in grape-like clusters (e.g. Staphylococcus spp.), and 10 were Gram-positive cocci chains (Enterococcus spp.).

[0124] Samples studied Two types of blood cultures were studied: (i)Bactec PLUS Aerobic / F, ref 442192(AR), and (ii) LYTIC / 10 ANAEROBIC / F ref 442265 (ANA) from Becton Dickinson, SA.

[0125] Both blood cultures were spiked (i.e. infected) with selected bacteria in the FASTinov laboratory in Porto (Portugal) according to the protocol described in [2] with minor modifications detailed below.

[0126] Bacterial suspensions were prepared by subculture of previously frozen isolates. Bacteria were grown overnight in Mueller-Hinton agar and the concentration was adjusted in phosphate-buffered saline (PBS). For this purpose, aerobic blood culture bottles were cultured with 2 × 10 bacteria per bottle along with 8 mL of whole blood sample obtained from a blood donor. 3 The inoculated blood cultures (BC) were incubated until a positive indicator on the recommended BD instrument. Blood culture bottles from BioMerieux were also tested. Urine samples from healthy donors were also inoculated with the same strains

[15] and subjected to the same protocol.

[0127] Extraction of microorganisms from samples To initiate AST using the novel FASTinov technology, purified bacterial suspensions obtained from clinical or veterinary samples were required. See Figure 1, two different protocols (Protocol A and Protocol B) for the extraction of bacteria from positive (i.e. infected) blood cultures were developed and the number of viable cells recovered was compared:

[0128] 1) Protocol A After vortexing of the blood cultures, vacutainer blood collection gel tubes (BD Vacutainer® Barricor™ blood collection tubes, ref 365056) were filled with approximately 5.5 ml and centrifuged at 1500 rpm for 5 min. The supernatant was discarded and the pellet was resuspended in 1 ml of sterile and filtered (through a 0.22 micrometer filter) saline. 1 ml of the suspension was transferred to an Eppendorf tube and centrifuged at 13,000×rpm (11337 g) for 1 min; the supernatant was then discarded. 1 ml of sterile and filtered (through a 0.22 micrometer filter) Muller-Hinton cation adjusted broth (ref BD 275730) was added to the Eppendorf tube and vortexed. Colony forming units (CFU) were subsequently assessed. This process was performed in duplicate.

[0129] 2) Protocol B After vigorous vortexing of the blood cultures, the cultures were loaded into vacuum blood collection gel tubes, e.g. 2 ml tubes (tube A in Figure 1), without any additives. A lysis agent, 50 μl of 10% (v / v) Triton X-100, was then added to the 1 ml contents of the collection tube, vortexed and incubated for 5 min at room temperature. The mixture was then centrifuged, preferably at 13000 rpm for 1 min, the supernatant discarded and the pellet resuspended in 0.5 ml of sterile and filtered (through a 0.22 micrometer filter) saline. Histopaque®-1077 (ref 10771) was added to a new tube (tube B in Figure 1) and the resuspended lysed sample was added to Histopaque; preferably 0.5 ml of the lysed sample was added to the 0.5 ml Histopaque®-1077 already in the tube and centrifugation was repeated, preferably at 13000 rpm for about 1 min; the supernatant was discarded. 1 ml of sterile and filtered (through a 0.22 micrometer filter) Mueller Hinton II cation adjusted broth (ref BD 275730) was added to the Eppendorf tube and vortexed. Colony forming units (CFU) were subsequently assessed. This process was performed in duplicate.

[0130] statistical analysis Differences between CFU obtained from aerobic (AR) and anaerobic (ANA) blood culture (BC) bottles after microbial extraction using protocol A, and from AR bottles, using protocols A and B, were assessed using the Wilcoxon signed rank test. All experiments were performed in duplicate and mean values ​​were calculated. Significant differences were considered at p-values ​​below 0.05 (<0.05). Statistical analysis was performed using IBM SPSS statistics version 24.0.

[0131] Identification of infectious agents The suspension obtained after treatment with TRITON X-100 and Histopaque was used to identify the microorganisms by MALDI-TOF (Bruker) using the septityper mode. Alternatively, samples purified via protocol B were further centrifuged at 13000-15000 rpm for 1 min. The resulting pellet was then dried at room temperature or at 37°C before inoculation into the MALDI-TOF (Bruker). The accuracy of detection obtained using the dried pellet was compared with that of overnight cultures.

[0132] (ii) Specifications of FASTinov Panel Optimization of different panel elements and processing times The inventors have designed an innovative panel that is used to assess the antimicrobial susceptibility of different types of bacteria to several antibiotics by flow cytometry. The optimization of antibiotic / probe combinations for selected groups of bacteria, such as Enterobacterales, Pseudomonas, Acinetobacter, Staphylococcus or Enterococcus, has resulted in the layout of a 96-well plate panel for Gram-positive bacteria (FASTgrampos - see Figures 4A and 4B) and a 96-well plate panel for Gram-negative bacteria (FASTgramneg - see Figures 3A and 3B). The inventors have very carefully selected the probes / concentrations / times that optimally separate susceptible from resistant strains among the wide variety of fluorescent probes available on the market.

[0133] For detection of some mechanisms of resistance, the Gram-negative panel includes some drugs such as ceftazidime-clavulanate and cefotaxime-clavulanate for ESBLs; cefoxitin for AmpC screening and low level meropenem (0.25ug / ml) to screen for carbapenemases.

[0134] The Gram-positive panel contains several concentrations of vancomycin, allowing both the MIC values ​​(minimum inhibitory concentrations) by flow cytometry and the phenotype to be determined.

[0135] Separate panels were designed to provide phenotypes and MICs for Enterobacteriales, Pseudomonas and Acinetobacter with respect to colistin susceptibility. The FASTcolistin MIC panel is shown in Figure 5A and Figure 5B. The antibiotic series and their order of appearance in the FASTgrampos and FASTgramneg panels were strategically developed by the inventors. Furthermore, the inventors implemented optimal washing between each well with the aim of reducing carryover effects identified during the development stage. If left uncontrolled, this carryover effect can generate adverse results, e.g., false susceptibility that would then induce major errors. Furthermore, two controls were included in the panel to ensure a valid susceptibility readout: (i) a positive control (untreated cells) that is commonly required in microbiology antimicrobial susceptibility assays, and (ii) a negative control (dead cells) to control for the viability of the probe, typically used in cytometry assays but not in microbiology assays. This novel addition confers an innovative twist to the new inventors' panel (see Figure 2).

[0136] Moreover, these controls ensure that only viable microorganisms are considered in the susceptibility assessment (and not non-viable ones). Non-viable microorganisms cannot respond to therapeutic agents. If they were included in the susceptibility assessment, the microorganisms could be mistakenly seen as resistant to all therapeutic agents, which would correspond to a false negative / diagnosis and a "major error" and, consequently, would result in the implementation of an incorrect therapy. Similarly, if the fluorescent probe is not present or does not function well, the effect of the therapeutic agent on the microorganisms is difficult or impossible to assess. Thus, the addition of a novel negative control significantly enhances the accuracy of the AST.

[0137] (iii) performing AST using the newly designed panel To perform AST using the new panel, we implemented the following steps: Microorganism Selection A substantial amount of ATCC bacteria was selected for this study. Fifteen Gram-negative rod-shaped bacteria were selected, including Escherichia coli ATCC 25922, Escherichia coli ATCC 8739, Escherichia coli ATCC 35218, Escherichia coli BAA 2425, Klebsiella pneumoniae ATCC 13443, Klebsiella pneumoniae BAA 1705, Klebsiella pneumoniae ATCC 700603, Klebsiella pneumoniae BAA1706, Enterobacter aerogenes ATCC 13048, Serratia marcescens ATCC 14756, Providencia rettgeri BAA 2525, Pseudomonas aeruginosa 27853, Pseudomonas aeruginosa BAA 2108, and Acinetobacter baumannii BAA1709. Ten additional Gram-positive cocci in grapevine in the grape-like cluster, including Staphylococcus aureus 29213, Staphylococcus aureus 43300, Staphylococcus aureus 700698, Staphylococcus epidermidis 35984; and five Gram-positive cocci chains, including Enterococcus faecalis 29212, Enterococcus faecalis 51299, Enterococcus faecium 700221, Enterococcus caceriflavus 700668, and Enterococcus gallinarum 49608, were selected. These bacteria represent the most important and common bacteria found in clinical situations. Tables 1, 2, and 3 below summarize the distribution of bacteria in each panel.

[0138] [Table 1]

[0139] [Table 2]

[0140] [Table 3]

[0141] Analyzed samples Blood cultures (Bactec PLUS Aerobic / F from Becton Dickinson, SA, ref 442192) were spiked with selected bacteria in the FASTinov laboratory in Porto (Portugal) according to the protocol described in [2] with minor modifications detailed above. Inoculated blood cultures (BC) were incubated until a positive indicator on the recommended BD instrument. Urine samples were also inoculated with the same strains and subjected to the same protocol, making urinary tract infection diagnosis a potential use of the newly designed panel for a large array of antibiotics.

[0142] Drug / fluorescent dye combinations The most important antimicrobial agents at breakpoint concentrations based on EUCAST and CLSI protocols were selected according to the microbial identification patterns for testing on different samples, preferably on blood cultures and urine samples prepared as detailed above (see Tables 4 and 5 below). Different concentrations of different fluorescent probes were tested; the lower concentration of each probe showing the best discrimination between untreated and dead cells (indicating high sensitivity) was selected.

[0143] [Table 4]

[0144] [Table 5]

[0145] Control Autofluorescence control wells (untreated and unstained cells), positive or viability control wells (untreated and stained cells with the respective fluorescent probe), and negative control wells (stained dead cells, which serve as a positive control for the fluorescent dye) were carefully distributed in the panel to ensure the viability of the strains and the activity of the probes. Different methods for negative control were tested at different concentrations, stained with different probes, and compared in flow cytometry analysis (see Figures 3-5).

[0146] A specific layout of panels The panel includes a first well for an autofluorescence control, followed by a positive control well stained with the probe without any antibiotic. Antibiotics were always placed on the panel from lower to higher concentration and combined with the probe. In the template of the flow cytometer acquisition, a 3-second backflush was scheduled between analyzed wells of the same drug; a 20-second backflush was programmed between wells of different drugs. Each well was mixed individually for 3 seconds. Drugs (e.g. antibiotics) that showed the highest intensity of fluorescence when incubated with the highly susceptible strain were intentionally placed at the edge of the panel. For example, given that the FASTgrampos panel has two different fluorescent probes, a second autofluorescent well and a control stained with the second probe were included before staining the drug with the second probe (see one of the panels displayed in Figures 3-5 for the panel layout). To confirm that the specificity of the fluorescent readout corresponds to actual marked cells and not to nonspecific or background fluorescent signals, a carryover study was undertaken (see Figures 3-5).

[0147] Dry Panel Performance and Controls All optimizations were performed on freshly manufactured panels, and lyophilization and drying processes were tested to obtain a room temperature stable product. In the room temperature stable samples, the antimicrobials were quantified using both microdilution assays and HPLC, and the fluorescent activity was evaluated by flow cytometry. Real-time stability studies were performed.

[0148] Inoculation and incubation of the panel Following sample preparation, a bacterial suspension with a MacFarland optical density of 0.5 was prepared in sterile saline solution and diluted in Mueller Hinton II cation adjusted broth (BD 275730). 6 ~1×10 7 A cell concentration of 1000 cells / ml was considered optimal. Cells were incubated for 1 h at 37° C. with shaking.

[0149] Flow cytometer analysis · For this analysis, the flow cytometer had the following specifications: it was equipped with one blue laser (488 nm; power: 50 mW; beam spot size: 5 × 80 μm); had three fluorescence channels: 525 / 40BP, 585 / 42BP, and 690 / 50BP; and Each panel was equipped with a plate reader for automated analysis.

[0150] Setting optimization was performed for Enterobacteriaceae, Acinetobacter, Pseudomonas, Staphylococcus and Enterococcus. Parameters such as cell fluorescence intensity, number of cells acquired by well and cell light scattering information were recorded and antibiotic treated cells were systematically compared to non-treated cells (positive control). Using our analytical (FASTinov) template, the panel was analyzed, data was recorded and reports were generated using bioFast software including proprietary algorithms. Validation criteria such as strain viability (positive control) and probe activity (negative control) were carefully included. Additionally, the number of events on the optimized gate (zone of the analysis where bacteria are presented) was also specifically included as a validation criterion. A minimum number of cells is required to validate the assay.

[0151] Reference method To evaluate the phenotypic results obtained from the flow cytometric analysis, antimicrobial susceptibility was determined for all isolates using the disk diffusion method as described in the reference protocols from EUCAST [3] and CLSI [4]. For colistin (in gram-negative bacteria) and vancomycin (for Staphylococcus), the method used to determine antimicrobial susceptibility was broth microdilution [5]. Broth microdilution was also performed for meropenem, since there was a need to know if the MIC was higher than 0.25ug / ml required for possible screening of carbapenemases [6]. Standard methodology was performed from the same inoculum used to spike the blood cultures. Quality control of the reference method was also performed according to the recommendations of EUCAST and CLSI.

[0152] Data analysis The highly susceptible (S), intermediate susceptible (I) and resistant (R) results obtained with the FASTinov® test were compared with MIC values ​​determined by reference disk diffusion and / or microdilution.

[0153] (iv) Performance evaluation Verification Facility Two facilities were used for validation of the different FASTinov® kits (FASTgrampos, FASTgramneg and FASTcolistinMIC) directly from positive blood cultures: the FASTinov laboratory in Porto (Portugal) using spiked blood cultures and the hospital Ramon y Cajal in Madrid (Spain) using patient blood cultures.

[0154] Bacterial strains A total of 256 Gram-negative bacilli (180 in FASTinov and 81 in Ramon y Cajal hospital) were studied in the FASTgramneg kit and 199 Gram-positive bacteria (131 in FASTinov and 68 in Ramon y Cajal hospital) in the FASTgrampos kit. Besides Enterobacteriaceae, Pseudomonas and Acinetobacter, different species of Staphylococcus and Enterococcus were studied. FASTcolistin MICs were performed both in FASTinov and in Ramon y Cajal hospital.

[0155] Antimicrobial susceptibility assays using reference methods All strains were classified as S, I or R according to the reference method. MICs were determined for vancomycin and colistin. Screening for ESBLs in Enterobacteriales group II according to the EUCAST protocol for detection of resistance mechanisms was also performed for the presence of ESBLs in Enterobacteriales group I, as well as for AmpC and the presence of carbapenemases.

[0156] Antimicrobial susceptibility assays using FASTinov kits (FASTgramneg, FASTgrampos and FASTcolistin MIC) Following the sample preparation protocol optimized by the inventors (FASTinov), i.e. protocol B for bacterial extraction from blood cultures as described above, inoculation of samples in the newly designed FASTinov panel and flow cytometric analysis, antimicrobial susceptibility results were obtained and compared with the reference method.

[0157] Data analysis Categorical agreement (CA) and essential agreement (EA) were calculated when applicable, as well as quantified error according to the standard definition of ISO 20776-2 (18). Error classification was performed as minor (mE), major (ME) and very major (VME). Screening for ESBLs and pAmpC in Enterobacteriales group I, proportion of agreement (PA), sensitivity and specificity for carbapenemases and ESBLs in Enterobacteriales group II were also performed. Disk diffusion or microdilution (in cases where disk diffusion assays are not recommended or there is a need to have a quantitative assay) was considered as the gold standard method and was used for comparison with the results of the FASTinov kit. Data analysis incorporated Expert Rules for Intrinsically Resistant Species to Certain Drugs (EUCAST) in the software.

[0158] (v) MALDI-TOF Identification Positive blood cultures of a total of 364 patients, 177 positive for Gram-positive cocci and 187 positive for Gram-negative bacilli, were processed to obtain bacterial suspensions according to the instructions and protocols for use of the FASTinov AST kit described above. However, for Maldi-TOF identification, the sample preparation step was carried out using Tergitol™ (Sigma) as follows:

[0159] A sterile microcentrifuge tube was filled with 1 ml of blood culture and mixed with 50 μL of Tergitol (10% v / v), followed by vortexing and centrifugation at 13000 rpm for 1 min. The supernatant was discarded and the resulting pellet was resuspended in 1 ml of sterile saline solution. The mixture was vortexed until the pellet was completely resuspended. 1 ml of this suspension was gently transferred onto the top of a microcentrifuge tube filled with 500 μL of Histopaque®-1077. The centrifugation was repeated, then the supernatant was discarded, and the resulting pellet was washed with saline solution, then centrifuged again and the supernatant was discarded. The wash-centrifuge-discard cycle was repeated using sterile water, and the final pellet was dried at 37°C for 5-10 min.

[0160] The final pellet was used directly on a MALDI-TOF target plate in two spots for each sample (i.e., in duplicate) using a wooden toothpick and 1 μl of α-cyano-4-hydroxycinnamic acid (CHCA) matrix added after the spots had dried. The target plate was placed in a Bruker MALDI Biotyper and the analysis was started using the Sepsityper sample type option on the instrument. Results were compared to colonies the next day and ID scores were recorded.

[0161] (vi) Validation studies Study design and sample collection The studies were carried out simultaneously at three sites. These included: First facility: FASTinov facility in Porto (Portugal) At this facility, the study used BACTEC blood bottles from Becton Dickinson (BD) that were spiked with ATCC strains from the American Type Culture Collection (ATCC) as well as well-characterized strains from the FASTinov bacterial collection according to the protocol described in reference 2 and incubated until a positive result for infection was recorded. · Second facility: the Hospital Ramon et Cajal in Madrid, a hospital with approximately 1000 beds. Sequential patient positive blood cultures (BACTEC, BD) were included in the study from November 2021 to February 2022 (one sample per patient). · Third facility: Centro Hospitalar S. Joao (CHSJ) in Porto, a university hospital with approximately 1000 beds. Sequential patient positive blood cultures (BACTEC, BD) were included in the study (one sample per patient). Blood cultures were collected from patients with suspected bloodstream infections from March 2022 to July 2022.

[0162] Positive blood cultures were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Germany) using the septityper mode. A wide variety of species was studied and are reported in Table 12. All tested strains were subcultured on blood agar panels to assess purity, and then reference susceptibility assays were performed on each strain, which were subsequently frozen at -80°C with approved study codification. Polymicrobial blood cultures were excluded from the study.

[0163] ethical considerations The study was approved by the Ethics Committee of the Ramon y Cajal University Hospital (reference number 161 / 17) and the Ethics Committee of the Centro Hospitalar S. Joao (reference number 284 / 21).

[0164] FASTinov assay Positive blood cultures previously identified by Maldi-Tof from each center were processed according to the Rapid AST of the FASTinov kit instructions for use as described above.

[0165] Analysis of cell damage To evaluate cell damage caused by antibiotics, flow cytometric analysis was performed using a Beckman Coulter CytoFlex model B3-R0-V3 (sites 1 and 2) and a DxFlex (site 3), both equipped with one blue laser (488 nm; power, 50 mW; beam spot size, 5×80 μm). The instruments have three fluorescent channels: 525 / 40BP, 585 / 42BP, and 690 / 50BP. The DxFlex is equipped with a plate reader for automated analysis of each panel. The flow cytometer was used in slow mode.

[0166] Software Analysis Proprietary software with algorithms defined by FASTinov was used for data analysis and results were considered in comparison with reference methods.

[0167] Instrument Timing The time that the panel spends in the instrument is the bottleneck of the assay, considering that the instrument can only analyze one panel at a time. Therefore, the time required for flow cytometric analysis of each panel depends only on the type of bacteria and the susceptibility protocol used (i.e. EUCAST or CLSI). Considering that the instrument is programmed to analyze in a fixed volume, acquisition in slow mode and automatically fixed washes between wells, this time is a fixed value assigned to the instrument.

[0168] reproducibility To include a range of phenotypes in each centre and to calculate inter-laboratory reproducibility, the 10 inoculated samples used in the FASTinov centre were also inoculated at each centre.

[0169] Reference method (RM) Positive blood cultures were spiked in blood agar, colonies identified and subjected to antimicrobial susceptibility assays by reference disk diffusion method and / or MIC values ​​determined by microdilution. Results were analyzed using the most recent EUCAST breakpoint tables and the M100 (29th edition) breakpoint tables from CLSI.

[0170] Example 1 Optimizing the sample preparation process Regarding sample preparation, Figures 1 and 2 show the number of CFU of cells recovered from aerobic (AR) and anaerobic (ANA) blood culture (BC) bottles using protocol A. For Gram-negative bacteria, the final number of organisms (CFU) obtained from AR and ANA bottles was not statistically different and was always above 1 × 10 8 / ml (>1×10 8 / ml).

[0171] Conversely, significant differences were observed for Staphylococcus spp., with the number of cells measured in the ANA bottles being above that measured in the AR bottles for 8 of the 10 different strains grown in the AR bottles. The number of Staphylococcus spp. cells recovered was significantly higher than that measured in the FASTinov panel (minimum 1 × 10 7 For Enterococcus spp., both the AR and ANA bottles contained enough cells (>1 × 10 6 cells / ml) to inoculate the FASTinov panel. 7 / ml), with slightly higher numbers measured in the AR bottles. The data presented in Table 6 below correspond to the average of the results obtained from two bottles per strain.

[0172] [Table 6]

[0173] Comparing protocols A and B, CFU were higher in all cases (Gram-negative bacilli and Gram-positive cocci) in protocol B, as seen in Table 7 below. This difference was even more significant for Staphylococci, considering that enough cells to perform the FASTinov assay could be obtained using protocol B and the AR BC bottles.

[0174] [Table 7]

[0175] Considering the results discussed above, and to simplify the method and provide a single sample preparation protocol that could work for all microorganisms and samples, protocol B was selected for all subsequent cases. As shown in Figure 1, protocol B involved the use of Triton X-100 and then subsequently Histopaque. The bacteria contained in the bacterial suspension obtained prior to preparing the subsequent suspension with a MacFarland optical density of 0.5 were accurately identified using MALDI-TOF Bruker.

[0176] Similar to what was observed with colonies from overnight blood cultures, dried samples purified via protocol B provided a high percentage of correct identification of microorganisms: 100% for Gram-negative bacteria (Enterobacteriales, Pseudomonas, and Acinetobacter) as shown in Figure 6A, and 98.3% for Gram-positive cocci (Staphylococcus and Enterococcus) as shown in Figure 6B.

[0177] This extraction protocol also produced excellent results (>10 6 CFU / ml).

[0178] Example 2 Selection of fluorescent probes The susceptibility results for each drug / antibiotic were compared to the reference method. Some combinations between drugs / fluorescent probes gave good results. Table 8 below summarizes the fluorescent probes that were tested in combination with the antibiotics disclosed in Tables 3 and 4.

[0179] [Table 8]

[0180] CTC (5-cyano-2,3-ditolyltetrazolium chloride, calcein AM, dihydrorhodamine 123, DIBAC4(3), DioC 2(3), fluorescein diacetate, 5CFDA, AM, CFDA-SE, propidium iodide, SYTO 16 green fluorescent, nucleic acid stain.

[0181] However, because each probe requires positive and negative controls, the number of fluorescent dyes selected was reduced to a minimum in order to reduce the number of controls. Additionally, the process of manufacturing development becomes more complicated when the number of probes in the panel is increased. Furthermore, the inventors needed to consider which probes would enable the panel to be stable at room temperature and the optimal drug / fluorescent dye combination. These processes were unique and complex, and therefore required extensive research and development.

[0182] For the FASTgramneg panel used to test for Gram-negative bacteria, DiBAC4(3) at 1 μg / ml was surprisingly identified as the probe to stain all drugs except imipenem in the case of Pseudomonas spp. Therefore, for this particular case, propidium iodide (PI) at 1 μg / ml gave better results (see FASTgramneg panel layout in Figure 3A and Figure 3B).

[0183] For the FASTgrampos panel used to test for Gram-positive bacteria, two fluorescent probes were selected depending on the drug of interest: PI at 1 μg / ml and DiOC2(3) (3,3′-diethyloxacarbocyanine iodide) at 0.06 uM (see FASTgrampos panel layout in Figure 4A and Figure 4B).

[0184] For the FASTcolistin MIC panel used to test for Gram-negative bacteria, 1 μg / ml PI was optimal (see FASTcolistin MIC layout in Figure 5A and Figure 5B). Untreated cells were stained with their fluorescent probes (positive control) and the highest test benzydamine concentration tested was selected for the negative control for all probes.

[0185] Example 3 Determination of bacterial susceptibility using the FASTinov method and associated kits The validation of the entire process, including the extraction protocol (Protocol B) and inoculation of the optimized panel, was evaluated together in all two laboratories, and the number and type of errors found in the CA, EA, and validation are shown in Table 9.

[0186] [Table 9]

[0187] [Table 10]

[0188] Example 4 MALDI-TOF identification The overall percent agreement (PA) of the FASTinov sample preparation method compared to the reference was approximately 95.6%. Detailed results are presented in Tables 10 and 11.

[0189] [Table 11]

[0190] [Table 12]

[0191] As can be seen in the table, no identification errors were observed, but in 16 cases out of a total of 364 no identification was obtained; the scores were very similar to colonies and divided by groups. The meaning of the score values ​​was defined according to the Bruker MALDI Biotyper using the cut-off values ​​of the Sepsityper sample type. The key findings from this study were as follows (higher scores correspond to better results): In Gram-positive cases, a score of <1.59 was observed in 14.3% of cases. Scores of 1.6 to 1.79 were observed in 4.1% of cases. A score of >1.8 was observed in 81.6%. In Gram-negative: the score is higher, i.e.: A score of <1.59 was observed in 15.6% of cases, Scores between 1.6 and 1.79 were not observed. A score of >1.8 was observed in 84.4%.

[0192] These results demonstrate that the FASTinov ultra-rapid AST sample preparation method can be used to perform MALDI-TOF identification and ultra-rapid AST in parallel for very time-efficient diagnosis while providing excellent accuracy for both Gram-positive and Gram-negative bacteria.

[0193] Example 5 Validation Study A total of 651 blood cultures from three centers were studied, with 348 isolates being gram-negative and 303 being gram-positive. The distribution of isolates by species per center is shown in Table 12.

[0194] [Table 13A]

[0195] [Table 13B]

[0196] Based on the analysis of results from a multicenter study using the EUCAST / CLSI guidelines, the sensitivity and specificity of the test were higher than 90%, as shown in Table 13. According to EUCAST / CLSI, the reproducibility for the FASTgramneg panel was 96.8% / 95% and for the FASTgrampos panel it was 95.1% / 95.1%.

[0197] [Table 14]

[0198] The time required to read the FASTinov panel by the flow cytometer software bioFAST depended on the number and concentration of drugs tested for each organism and on the protocol selected. The recorded time values ​​are reported in Table 14 below. The minimum time was 9 minutes for Acinetobacter species with the EUCAST protocol and the maximum was 47 minutes for Enterobacteriaceae according to the CLSI protocol.

[0199] [Table 15]

[0200] Very Major Result (VME) Details of the VME are presented in Table 15. Higher numbers of strains were observed for piperacillin / tazobactam and ceftolozane / tazobactam strains (four each). CA was 100% for amoxicillin / clavulanic acid (EUCAST) and ceftazidime / avibactam and amikacin (both EUCAST / CLSI).

[0201] [Table 16]

[0202] Results observed with the FASTgramneg kit The overall categorical agreement (CA) of the FASTgramneg kit was >95% with an error of <1.5%.

[0203] Fifty-three of the Enterobacteriales group I strains were ESBL positive in the reference method (RM), and the FASTinov assay had a sensitivity and specificity of 96.2% and 100%, respectively, with a PA of 99.0%. For screening for ESBL in group II Enterobacteriales bacteria, 13 were positive, with a sensitivity, specificity of 100%, and a PA of 100%. For plasmid AmpC screening (Enterobacteriales group I), 38 were positive in the RM, with a concordance (PA) of 100% as well as sensitivity and specificity. Fifty-two of the strains were positive for carbapenemase (MIC > 0.25ug / ml for meropenem), and the FASTinov test showed a sensitivity of 96.2% (two false negatives were found in strains with a MIC of 0.5ug / ml) and a specificity of 96.7%, with a PA of 96.6%.

[0204] [Table 17]

[0205] FASTgrampos Kit Gram-positive kits analyzed according to EUCAST guidelines achieved >95% sensitivity and specificity and >95% CA (Table 17). All tested drugs showed >90% CA. ME and mE were also low.

[0206] [Table 18]

[0207] Essential agreement (EA) for MIC determination for vancomycin in S. aureus was 100% with a bias of approximately 30%, which is the lower limit recommended in ISO 20776-2:2021.

[0208] Table 18 shows the distribution of MICs for the S. aureus strains tested. The FASTinov method provides one dilution advantage over the standard method in many cases.

[0209] [Table 19]

[0210] The results obtained at each individual center are provided below in Tables 19-24 (Tables 20-25).

[0211] [Table 20]

[0212] [Table 21]

[0213] [Table 22]

[0214] [Table 23]

[0215] [Table 24]

[0216] [Table 25]

[0217] Observations and Conclusions Rapid antimicrobial assays are urgently needed in hospitals, especially in critical situations, such as sepsis. The inventors have developed a highly innovative and groundbreaking technique of antimicrobial susceptibility testing (AST) evaluation together with an improved protocol for efficient extraction of microorganisms from clinical samples at concentrations that allow them to be easily analyzed by flow cytometry. 7 A clean (with as little debris as possible) suspension of microorganisms, at a concentration of 10000 / ml, was required to perform the new and rapid AST assay. We started to extract microorganisms (bacteria or fungi) from positive blood or urine cultures or any other biological samples using barricot tubes (protocol A above). In the process, some of the bacterial cells were lost during centrifugation (the bacterial cells formed a pellet together with the human / animal cells and debris at the bottom of the tube), but in most cases a suspension with sufficient cells could be obtained. The most problematic case was observed with Staphylococcus in aerobic blood cultures, possibly due to its usual conformation in liquid media (grape-like clusters would go to the bottom in centrifugation). However, this situation does not occur in anaerobic blood cultures, probably because they contain saponin, which allows the formation of large grape-like clusters during the division process.

[0218] With the knowledge that Staphylococcus is one of the most common microorganisms recovered from blood cultures in clinical settings and that anaerobic blood bottles are the most common blood cultures in laboratories, the inventors have developed an improved extraction method that has proven highly effective for the extraction of microorganisms, and particularly Staphylococcus, namely Protocol B described above and shown in FIG. 1.

[0219] Histopaque®-1077 can create a concentration gradient of blood components and is particularly used to separate white blood cells (top of Histopaque®-1077) from red blood cells (bottom). If the blood sample contains microorganisms, they will go to the bottom with the red blood cells. Using lysed blood culture samples (treated with a lysing agent such as Triton X-100 or Tergitol) before adding the suspension to Histopaque®-1077, a clean and concentrated suspension of microorganisms was obtained after centrifugation on Histopaque®-1077. Protocol B is particularly useful when the microorganism that needs to be extracted is Staphylococcus. However, it can be used with both aerobic or anaerobic blood cultures in bottles (BD or BioMerieux) and, surprisingly, even with other biological samples such as urine, bronchial secretions, and cerebrospinal fluid, when one wishes to extract any and all microbial species. This is therefore the first report of the novel extraction protocol developed by the inventors.

[0220] For the panel (i.e. kit) developed by the inventors, several fluorescent probes could be used for each antimicrobial agent. However, the inventors decided to use the same probe for most cases when possible, because each probe used requires a corresponding positive and negative control. Therefore, if several probes were included, this would negatively affect and prolong the time required to perform the assay and to generate an antibiotic susceptibility reading. Furthermore, the process of drying each probe is complicated, and therefore the inventors chose to include as few probes as possible.

[0221] Three controls were designed for this assay. First, a positive control, which is a control classically used in microbiology assays to ensure the viability of the strain. Two other controls, autofluorescence and a negative control, are typically used for cytometry assays to verify whether the stain is present (i.e., the difference between stained and unstained cells) and that the probe stains the cells properly. This is the first report of their use in a microbiology assay.

[0222] In summary, the assay therefore includes a positive control to confirm bacterial survival. The positive control is achieved with bacterial cells that are not treated with antibiotics but exposed to a fluorescent stain that was confirmed during development to not affect bacterial cell survival. In the autofluorescence control, the bacterial cells are not treated with antibiotics and are not stained with the fluorescent probe. In the negative control, the bacterial cells are exposed to a killing agent and stained with the fluorescent probe. This is key to control the quality of the fluorescent compound to ensure that the fluorescent agent marks the bacteria if they are damaged or even dead.

[0223] Also, this is the first report of the innovative layout of the panel designed by the inventors. However, it is understood that the reported panel layout may be modified within the scope of the present invention for several reasons, such as the introduction of new drugs, changes in CLSI and EUCAST breakpoints, the introduction of different fluorescent dyes, or even changes in the series of drugs.

[0224] The striking results for CA, EA and a small number of VME and ME reported in Table 9 are in line with FDA and ISO 20776-2 recommendations and demonstrate that the methods, assays and kits described herein have the potential to revolutionize antimicrobial therapeutic approaches worldwide, with significant clinical and public health impact.

[0225] As shown in a multicenter study, the FASTinov® kit consistently provides ultra-rapid AST (<2 hours) with high accuracy and reproducibility, resulting in improved and timely diagnosis in septic patients, thereby impacting their clinical outcomes. (References) TIFF2025508599000028.tif198148

Claims

1. 1. A method for determining the susceptibility phenotype of a microorganism present in a biological sample to at least one therapeutic agent, comprising: (i) a biological sample containing a microorganism, (a) one or more first test reservoirs; (b) one or more second positive control reservoirs; (c) one or more third negative control reservoirs containing non-viable microorganisms; and (d) one or more fourth autofluorescent control reservoirs that contain neither the therapeutic agent nor the fluorescent marker. introducing into each of (ii) contacting the biological sample in the one or more first test reservoirs with at least one therapeutic agent; (iii) contacting the biological sample in the one or more first, second, and third reservoirs with at least one fluorescent marker; and (iv) performing a fluorescence analysis to obtain one or more fluorescence parameters for the biological sample in each of the reservoirs. Including, a susceptibility phenotype of the microorganism to the at least one therapeutic agent is obtained by comparing one or more fluorescence parameters between the reservoirs; method.

2. the one or more first test reservoirs contain viable or living microorganisms, optionally at least 60%, 70%, or 80% viable cells; and / or 10. The method of claim 1, wherein the one or more second positive control reservoirs contain live or living microorganisms, optionally at least 60%, 70%, or 80% viable cells.

3. 2. The method of claim 1, wherein the one or more third negative control reservoirs are made non-viable by exposure to a cell killing agent, optionally wherein the one or more third negative control reservoirs contain the cell killing agent, optionally wherein the one or more third negative control reservoirs contain at least 60%, 70%, or 80% non-viable cells, optionally wherein the cell killing agent is selected from the group consisting of ethanol, 2-phenoxyethanol, citric acid, and benzydamine hydrochloride, optionally wherein the cell killing agent is benzydamine hydrochloride.

4. The method of claim 1, wherein the susceptibility phenotype is highly susceptible, intermediately susceptible, or resistant.

5. 10. The method of claim 1, wherein the one or more fourth autofluorescence control reservoirs contain live or living microorganisms, optionally at least 60%, 70%, or 80% viable cells.

6. 10. The method of claim 1, further comprising a sample preparation step, optionally comprising purifying the microorganism from the biological sample before the biological sample is introduced into each of the first, second, third and / or optionally, fourth reservoirs.

7. The sample preparation step (i) obtaining said biological sample containing said microorganism; and (ii) contacting the sample with a density gradient solution Including, 7. The method of claim 6, wherein optionally the density gradient solution is histopaque®, and optionally the sample preparation step comprises contacting the biological sample with a hemolyzing agent prior to contacting the biological sample with the density gradient solution, and optionally the hemolyzing agent is Triton X-100 or Tergitol.

8. The sample preparation step (iii) identifying the microorganism prior to introducing the biological sample containing the microorganism into each of the one or more first, second, third and / or optionally fourth reservoirs. Further comprising:

7. The method of claim 6, wherein optionally, the identification of the microorganism is performed using a mass spectrometry method, preferably MALDI-TOF mass spectrometry, and optionally, the biological sample is dried prior to the identification of the microorganism, and optionally, drying of the biological sample is achieved by a centrifugation step and / or an air drying process.

9. 2. The method of claim 1, wherein the biological sample is of human, animal or environmental origin, preferably wherein the sample is of human origin, and optionally wherein the biological sample of human origin comprises tissue, blood, plasma, serum, spinal fluid, urine, bronchial secretions, cerebrospinal fluid, sweat, saliva, sputum, tears, breast aspirate, prostatic fluid, semen, vaginal fluid, feces, cervical scraping, amniotic fluid, intraocular fluid, mucus, breath moisture, animal tissue, cell lysate, tumor tissue, hair, skin, buccal scrapings, nail, bone marrow, cartilage, prion, bone powder, earwax, or a combination thereof.

10. the microorganism is selected from the group consisting of a bacterium, a virus, a fungus or a protozoan, preferably the microorganism is a bacterium, and / or 10. The method of claim 1, wherein the therapeutic agent is selected from the group consisting of an antibiotic, an antiviral, an antifungal; and an antiprotozoan, preferably the therapeutic agent is an antibiotic.

11. The method of claim 1, wherein the fluorescence analysis is flow cytometry analysis or laser scanning analysis, preferably the fluorescence analysis is flow cytometry analysis.

12. The method of claim 1, wherein (i) the one or more flow cytometry parameters include forward scatter and / or side scatter and / or fluorescence parameters, and optionally the fluorescence scatter signal is intensity, spectral profile and / or cell count; (ii) the fluorescent marker is selected from the group consisting of acridine dyes; cyanine dyes; fluorone dyes; oxazine dyes; phenanthridine dyes; and rhodamine dyes; (iii) the fluorescent marker is selected from the group consisting of CTC (5-cyano-2,3-ditolyltetrazolium chloride), calcein AM, dihydrorhodamine 123, DIBAC4(3), DioC 2(3), fluorescein diacetate, 5CFDA, AM, CFDA-SE, propidium iodide, SYTO 16 green fluorescence, and nucleic acid stains; and / or (iv) The method of claim 1, wherein the reservoir is a container, a tube or a well, preferably the reservoir is a well.

13. 13. A kit for use in the method of any one of claims 1 to 12.

14. 14. The kit of claim 13, wherein the kit is a panel comprising the one or more first, second, third, and fourth reservoirs, optionally the panel being a 96-well plate.

15. the panel is configured for analyzing Gram-negative bacteria, and optionally the panel is substantially as shown in FIG. 3A or FIG. 5A; or 15. The kit of claim 14, wherein the panel is configured to analyze Gram-positive bacteria, and optionally the panel is substantially as shown in FIG. 4A.

Images