Methods and systems for the detection and removal of pathogens from blood

Abstract

The invention relates to methods and systems for removal of pathogens from blood or blood products. The invention further relates to methods and systems for treatment and diagnosis of infection in the blood and/or sepsis in a patient in need thereof.

1 . A method for detecting pathogens selected from the group consisting of a bacterium, a virus, and a fungus in a sample of blood or of a blood product comprising red blood cells (RBCs), the method comprising:
 i) contacting said sample with a leukocyte reduction filter or with a support selected from the group consisting of a filter, microfibers, microparticles, beads, microspheres, an array, glass slides and microscope slides, said support comprising a non-specific RBC adhesion molecule binder, wherein said non-specific RBC adhesion molecule binder is a polymer or a mixture of two or more polymers that binds to adhesion molecules that become activated and are expressed on RBCs and is selected from one or more of the group consisting of polyester, polyurethane, cellulose, nylon, nitrocellulose, polypropylene, polyethylene terephthalate (PET), polyimide (PI), polyvinyl alcohol (PVA), and polyvinylidene fluoride (PVDF), and mixtures thereof, to allow binding of RBCs that have bound at least one pathogen to said leukocyte reduction filter or said support;   ii) detecting the presence of RBCs that have bound at least one pathogen.   
     
     
         2 . The method according to  claim 1  further comprising separating said sample into at least two components prior to step i), a first component comprising red blood cells (RBCs) and substantially free of white blood cells (WBCs) and platelets and a second component comprising WBCs and platelets and substantially free of RBCs. 
     
     
         3 . The method according to  claim 1  further comprising isolating RBCs that are bound to said leukocyte reduction filter or said support from said sample, preferably by removing cells and other components in said sample that have not bound to said leukocyte reduction filter or said support. 
     
     
         4 . The method according to  claim 3 , further comprising lysing said RBCs that are bound to leukocyte reduction filter or said support following said isolation step. 
     
     
         5 . The method according to  claim 1 , further comprising determining at least one characteristic of said pathogens if RBCs that have bound at least one pathogen are detected in said sample, wherein said characteristic is selected from the group consisting of the type of pathogen, gram-positivity or gram-negativity if said pathogen is a bacterium, drug or antibiotic resistance of the pathogens, and the identity of the pathogens. 
     
     
         6 . The method according to  claim 1 , wherein said support comprises beads coated with said non-specific RBC adhesion molecule binder. 
     
     
         7 . The method according to  claim 1  wherein said sample is a sample from an individual having an infectious pathogen in the blood, an individual suspected of having an infectious pathogen in the blood, an individual suffering from sepsis, an individual suspected of suffering from sepsis, an individual at risk of suffering from sepsis. 
     
     
         8 . The method according to  claim 1 , further comprising identifying pathogens in the sample of blood or the blood product comprising red blood cells (RBCs), the method comprising isolating RBCs that are bound to said leukocyte reduction filter or said support from said sample, preferably by removing cells and other components in said sample that have not bound to said leukocyte reduction filter or said support; and identifying the pathogen that is bound to said RBCs bound to said leukocyte reduction filter or said support. 
     
     
         9 . The method according to  claim 1  for diagnosing an infection in the blood of a patient. 
     
     
         10 . The method according to  claim 1  for isolating and/or enriching pathogens selected from the group consisting of a bacterium, a virus, and a fungus from a sample of blood or of a blood product comprising red blood cells (RBCs), the method further comprising isolating RBCs that are bound to said leukocyte reduction filter or said support from said sample, preferably by removing cells and other components in said sample that have not bound to said leukocyte reduction filter or said support. 
     
     
         11 . The method according to  claim 1  for determining susceptibility for antibiotics of a pathogen, the method comprising:
 isolating RBCs that are bound to said leukocyte reduction filter or said support from said sample; 
 contacting said isolated RBCs that are bound to said leukocyte reduction filter or said support with one or more antibiotic agents; and 
 determining the growth or a functional activity of the pathogen. 
 
     
     
         12 . A system ( 1 ) for removing a pathogen from blood or a blood product, comprising:
 a separation device ( 2 ), preferably comprising a centrifuge, for separating said blood or said blood product into at least a first component comprising red blood cells (RBCs) and being substantially free of white blood cells (WBCs) and platelets and a second component comprising white blood cells (WBCs) and platelets and being substantially free of red blood cells (RBCs);   a filter device ( 3 ) for depleting said first component substantially of RBCs that have bound at least one pathogen, said filter device comprising a non-specific RBC adhesion molecule binder, wherein said non-specific RBC adhesion molecule binder preferably is a polymer;   a conduit ( 4 ) for conducting said first component from the separation device ( 2 ), e.g. from a first outlet thereof ( 2   b ), to the filter device ( 3 ), e.g. to an inlet thereof ( 3   a );   an outlet conduit ( 10 ) for conducting the filtered first component, which is substantially depleted of RBCs that have bound at least one pathogen, away from said filter device ( 3 ), wherein the system ( 1 ) is arranged to feed the filtered first component, which is substantially depleted of RBCs that have bound at least one pathogen, to a subject ( 9 ) and/or to an administering device ( 6   b ) for administering at least said filtered first component to a subject ( 9 ).   
     
     
         13 . A system ( 1 ) according to  claim 13  further comprising:
 a second component outlet conduit ( 11 ) for conducting the separated second component away from the separation device ( 2 ), said second component outlet conduit ( 11 ) being in fluid connection with a second outlet ( 2   c ) of the separation device ( 2 ) for letting the second component out of the separation device ( 2 ), wherein the system ( 1 ) is arranged to put the filtered first component and at least part of the second component of the blood or blood product together in order to obtain a blood or blood product substantially free of pathogens, or at least substantially free of RBC bound pathogens, preferably wherein the outlet conduit ( 10 ) for conducting the filtered first component away from the filter device ( 3 ) merges with the second component outlet conduit ( 11 ), such that the filtered first component and the second component can join, and can preferably mix. 
 
     
     
         14 . A system ( 1 ) according to  claim 13 , further comprising a mixing chamber ( 12 ) or another mixing unit ( 12 ) for mixing said second component with said filtered first component. 
     
     
         15 . A system ( 1 ) according to  claim 12 , further comprising an anticoagulant supply unit ( 8 ) for adding an anticoagulant to the blood or the blood product, wherein said anticoagulant supply unit ( 8 ) is preferably situated upstream of the separation device ( 2 ), and more preferably is arranged for feeding the anticoagulant into a supply conduit ( 5 ) for supplying the blood or blood product to the separation device ( 2 ). 
     
     
         16 . An apparatus for removing a pathogen from blood or a blood product comprising a housing ( 15 ) and a system ( 1 ) according to  claim 12 . 
     
     
         17 . A method for determining the presence of pathogens in a whole blood sample of an individual suspected of having an infectious pathogen in the blood, suffering from sepsis, suspected of suffering from sepsis or at risk of developing sepsis, the method comprising detecting the presence of red blood cells (RBCs) that have bound at least one pathogen using cytometry or microscopy. 
     
     
         18 . The method according to  claim 17 , wherein said detecting step is carried out by flow cytometry whereby RBCs that have bound at least one pathogen are detected by cell sorting for RBCs and labelling of pathogens. 
     
     
         19 . The method according to  claim 18 , wherein said labelling of pathogens comprises DNA labelling. 
     
     
         20 . The method according to  claim 18 , wherein the method comprises determining the presence of bacteria and said labelling comprises labelling with a general bacteria label. 
     
     
         21 . The method according to  claim 17 , wherein said individual is suspected of having an infectious pathogen in the blood, suspected of suffering from sepsis or at risk of developing sepsis.