In vitro process for the quick determaination of a patient's status relating to infection with mycobacterium tuberculosis

Abstract

An in-vitro process for the quick determination of the infection status of a Mycobacterium tuberculosis infection from whole blood in terms of active or latent tuberculosis , comprising the steps of: stimulating an antigen-specific T cell that is present in a first sample of whole blood with purified protein derivative (PPD) in the presence of antibodies against CD28, or CD28 and CD49d; processing of PPD by antigen-presenting cells (APC) by incubating for 1.5 h to 2.5 h, especially for 2 h, at 35° C. to 39° C., especially at 37° C., optionally with adding CO 2 ; then adding a secretion inhibitor; effecting an intensive mixing; and another incubation for a period of at least 2.5 h at a temperature of from 35° C. to 39° C., and determining a cytokine profile from both the intracellular INF-γ production and the intracellular IL-2 production of the antigen-specific T cell; wherein the presence of an active tuberculosis is indicated by a shift of the cytokine profile towards IFN-γ single positive cells accompanied by a decrease of IFN-γ/IL-2 double positive T cells.

1 . An in-vitro process for the quick determination of the infection status of a  Mycobacterium tuberculosis  infection from whole blood in terms of an active or latent  tuberculosis,  comprising the steps of:
 stimulating an antigen-specific T cell that is present in a first sample of whole blood with purified protein derivative (PPD) in the presence of antibodies against CD28, or CD28 and CD49d;   processing of PPD by antigen-presenting cells (APC) by incubating for 1.5 h to 2.5 h at 35° C. to 39° C. optionally with adding CO 2 ;   then adding a secretion inhibitor;   effecting an intensive mixing; and   another incubation for a period of at least 2.5 h at a temperature of from 35° C. to 39° C., and   determining a cytokine profile from both the intracellular INF-γ production and the intracellular IL-2 production of the antigen-specific T cell; wherein   the presence of an active  tuberculosis  is indicated by a shift of the cytokine profile towards IFN-γ single positive cells accompanied by a decrease of IFN-γ/IL-2 double positive T cells.   
     
     
         2 . The process according to  claim 1 , wherein another stimulation with SEB, ESAT-6 and CFP-10 and/or vaccination antigen is effected in addition to said stimulation with PPD. 
     
     
         3 . The process according to  claim 1 , wherein a negative control is performed by adding a physiological buffer such as PBS 0.9% NaCl or 5% Glucose to an antigen-specific T cell that is present in the whole blood of a second sample, which is authentic with said first sample. 
     
     
         4 . The process according to  claim 1 , wherein a positive control is performed by adding SEB to an antigen-specific T cell that is present in the whole blood of a third sample, which is authentic with said first sample. 
     
     
         5 . The process according to  claim 1  wherein a vaccination control took place by adding the vaccination antigen to an antigen-specific T-cell that is present in the whole blood of a fourth sample which is authentic with said first sample. 
     
     
         6 . The process according to  claim 1 , wherein a BCG-vaccination control is effected by adding ESAT-6 (early secreted antigenic target) and CFP-10 (culture filtrate protein) to an antigen-specific T cell that is present in the whole blood of a fourth sample, which is authentic with said first sample. 
     
     
         7 . The process according to  claim 1  wherein a control whether or not a contact with non- tuberculosis  mycobacteria took place in the whole blood donor is effected by adding ESAT-6 (early secreted antigenic target) and CFP-10 (culture filtrate protein) to an antigen-specific T cell that is present in the whole blood of a fifth sample, which is authentic with said first sample. 
     
     
         8 . The process according to  claim 1 , wherein said secretion inhibitor is selected from the group consisting of brefeldin A, monensin or the like. 
     
     
         9 . The process according to  claim 1 , wherein the determination of the cytokine profile is effected by flow cytometry. 
     
     
         10 . The process according to  claim 1 , wherein the whole blood of the samples is derived from humans. 
     
     
         11 . The process according to  claim 1  applied for contact tracing wherein whole blood was obtained from a person with close contact to a patient with active TB or suspect to have had a close contact to a patient with active TB. 
     
     
         12 . The process according to  claim 11  wherein a treatment decision is made as follows:
 a) a prophylaxis or an active therapy is indicated in cases, where a shift of the cytokine profile towards IFN-γ single positive cells accompanied by a decrease of IFN-γ/IL-2 double positive cells was detected; 
 b1) if diagnostic investigations revealed a latent infection with  Mycobacterium tuberculosis  by IFN-γ/IL-2 double positive cells and no information about previous testing is available, no acute therapy is necessary and the patient has to be retested within 2 to 4 weeks; 
 b2) if diagnostic investigations revealed a latent infection with  Mycobacterium tuberculosis  by IFN-γ/IL-2 double positive cells and if in previous testing for latent infection with  Mycobacterium tuberculosis  did reveal no contact with  Mycobacterium tuberculosis  so far, a prophylaxis is indicated; 
 c1) no acute therapy is indicated if the patient shows no response; 
 c2) in case the patient reveals any clinical symptoms, retesting is indicated. 
 
     
     
         13 . The process according to  claims 1  wherein whole blood was obtained from a person before receiving immunosuppressive drugs or in an immune-incompetent state like HIV infection. 
     
     
         14 . The process of  claim 13  wherein a treatment decision is made as follows:
 a) a prophylaxis or an active treatment, and optionally delaying of immunosuppressive therapy, is indicated in cases where a shift of the cytokine profile towards IFN-γ single positive cells accompanied by a decrease of IFN-γ/IL-2 double positive cells was detected; 
 b1) if diagnostic investigations revealed a latent infection with  Mycobacterium tuberculosis  by IFN-γ/IL-2 double positive cells and no information about previous testing is available, immunosuppressive therapy is indicated and the patient has to be retested within 2 to 4 weeks; 
 b2) if diagnostic investigations revealed a latent infection with  Mycobacterium tuberculosis  by IFN-γ/IL-2 double positive cells and if in previous testing for latent infection with  Mycobacterium tuberculosis  did reveal no contact with  Mycobacterium tuberculosis  so far, a prophylaxis and optionally delaying of immunosuppressive therapy, is indicated as well as retesting of the patient within 2 to 4 weeks; 
 c1) immunosuppressive therapy is to begin if the patient shows no response; 
 c2) in case the patient reveals any clinical symptoms, retesting is indicated. 
 
     
     
         15 . The process according to  claims 1 , wherein whole blood was obtained from a person suspected for an active TB infection. 
     
     
         16 . The process of  claim 15  wherein a treatment decision is made as follows:
 a) an active treatment is indicated in cases where a shift of the cytokine profile towards IFN-γ single positive cells accompanied by a decrease of IFN-γ/IL-2 double positive cells was detected; 
 b1) if diagnostic investigations revealed an latent infection with  Mycobacterium tuberculosis  by IFN-γ/IL-2 double positive cells and no information about previous testing is available, an acute infection is unlikely, an alternative diagnosis is to be considered, and the patient has to be retested within 2 to 4 weeks; 
 b2) If diagnostic investigations revealed a latent infection with  Mycobacterium tuberculosis  by IFN-γ/IL-2 double positive cells and if in previous testing for latent infection with  Mycobacterium tuberculosis  did reveal no contact with  Mycobacterium tuberculosis  so far, a prophylaxis or an active treatment is indicated and the patient has to be retested within 2 to 4 weeks; 
 c1) no acute therapy is indicated if the patient shows no response; 
 c2) in case the patient reveals any clinical symptoms, retesting is indicated. 
 
     
     
         17 . The process according to  claim 1 , wherein processing of PPD by antigen-presenting cells (APC) is by incubating for 2 h at 37° C., optionally with adding CO2. 
     
     
         18 . The process according to  claim 1 , wherein a negative control is performed by adding a physiological buffer selected from the group consisting of PBS 0.9% NaCl and 5% Glucose to an antigen-specific T cell that is present in the whole blood of a second sample, which is authentic with said first sample. 
     
     
         19 . The process according to  claim 1 , wherein the whole blood of the samples is derived from animals. 
     
     
         20 . The process according to  claim 11  wherein a treatment decision is made as follows:
 a) INH prophylaxis or quadruple therapy is indicated in cases where a shift of the cytokine profile towards IFN-γ single positive cells accompanied by a decrease of IFN-γ/IL-2 double positive cells was detected; 
 b1) if diagnostic investigations revealed a latent infection with  Mycobacterium tuberculosis  by IFN-γ/IL-2 double positive cells and no information about previous testing is available, no acute therapy is necessary and the patient has to be retested within 2 to 4 weeks; 
 b2) if diagnostic investigations revealed a latent infection with  Mycobacterium tuberculosis  by IFN-γ/IL-2 double positive cells and if in previous testing for latent infection with  Mycobacterium tuberculosis  did reveal no contact with  Mycobacterium tuberculosis  so far, INH prophylaxis is indicated; 
 c1) no acute therapy is indicated if the patient shows no response; 
 c2) in case the patient reveals any clinical symptoms, retesting is indicated. 
 
     
     
         21 . The process of  claim 13  wherein a treatment decision is made as follows:
 a) INH prophylaxis or quadruple therapy, and optionally delaying of immunosuppressive therapy, is indicated in cases where a shift of the cytokine profile towards IFN-γ single positive cells accompanied by a decrease of IFN-γ/IL-2 double positive cells was detected; 
 b1) if diagnostic investigations revealed a latent infection with  Mycobacterium tuberculosis  by IFN-γ/IL-2 double positive cells and no information about previous testing is available, immunosuppressive therapy is possible and the patient has to be retested within 2 to 4 weeks; 
 b2) if diagnostic investigations revealed a latent infection with  Mycobacterium tuberculosis  by IFN-γ/IL-2 double positive cells and if in previous testing for latent infection with  Mycobacterium tuberculosis  did reveal no contact with  Mycobacterium tuberculosis  so far, INH prophylaxis, and optionally delaying of immunosuppressive therapy, is indicated as well as retesting of the patient within 2 to 4 weeks; 
 c1) immunosuppressive therapy is to begin if the patient shows no response; 
 c2) in case the patient reveals any clinical symptoms, retesting is indicated. 
 
     
     
         22 . The process of  claim 15  wherein a treatment decision is made as follows:
 a) quadruple therapy is indicated in cases where a shift of the cytokine profile towards IFN-γ single positive cells accompanied by a decrease of IFN-γ/IL-2 double positive cells was detected; 
 b1) if diagnostic investigations revealed an latent infection with  Mycobacterium tuberculosis  by IFN-γ/IL-2 double positive cells and no information about previous testing is available, an acute infection is unlikely, an alternative diagnosis is to be considered, and the patient has to be retested within 2 to 4 weeks; 
 b2) If diagnostic investigations revealed a latent infection with  Mycobacterium tuberculosis  by IFN-γ/IL-2 double positive cells and if in previous testing for latent infection with  Mycobacterium tuberculosis  did reveal no contact with  Mycobacterium tuberculosis  so far, INH prophylaxis or quadruple therapy is indicated and the patient has to be retested within 2 to 4 weeks; 
 c1) no acute therapy is indicated if the patient shows no response; 
 c2) in case the patient reveals any clinical symptoms, retesting is indicated.