Distinguishing cells in a sample by inactivating extracellular enzyme before releasing intracellular enzyme

Abstract

A method for detecting the absence or presence of cells of interest in a liquid sample, wherein: (a) the sample: (i) comprises an extracellular medium containing an enzyme with a measurable activity; and (ii) is suspected of containing cells of interest that contain an enzyme with said measurable activity; and (b) the method comprises the steps of: (i) treating the liquid sample with a reagent that inactivates said measurable activity in the extracellular medium, but does not inactivate the measurable activity in said cells of interest; (ii) lysing the cells of interest to release the intracellular enzyme; and (iii) measuring said measurable activity. Thus the intracellular enzyme can be measured without interference from the extracellular enzyme. The invention is particularly useful for treatment of bacterially-infected blood using a detection assay based on adenylate kinase activity.

1 . A method for detecting the absence or presence of cells of interest in a liquid sample, the method comprising:
 treating a liquid sample comprising an extracellular medium;
 wherein the extracellular medium contains an extracellular enzyme having a measurable enzymatic activity; 
 wherein the sample is suspected of comprising cells of interest that comprise an intracellular enzyme having the measurable enzymatic activity; 
 wherein treating the liquid sample comprises treating the sample with a reagent that inactivates the measurable enzymatic activity in the extracellular medium, but does not inactivate the measurable enzymatic activity in the cells of interest; 
   lysing said cells of interest to release the intracellular enzyme; and   measuring the measurable enzymatic activity.   
     
     
         2 . The method of  claim 1 , wherein the cells of interest are microorganism cells. 
     
     
         3 . The method of  claim 2 , wherein the microorganism cells comprise bacterial cells. 
     
     
         4 . The method of  claim 1 , wherein the liquid sample comprises a blood sample. 
     
     
         5 . The method of  claim 4 , wherein the blood sample comprises lysed blood cells and unlysed microbial cells. 
     
     
         6 . The method of  claim 1 , wherein the measurable enzymatic activity comprises adenylate kinase activity or lactate dehydrogenase activity. 
     
     
         7 . The method of  claim 6 , wherein the adenylate kinase activity is measured via a luciferase/luciferin reaction. 
     
     
         8 . The method of  claim 1 , wherein the reagent comprises a protease. 
     
     
         9 . The method of  claim 8 , wherein the protease comprises trypsin. 
     
     
         10 . A method comprising:
 treating a liquid sample comprising an extracellular medium;
 wherein the extracellular medium contains an extracellular enzyme having a measurable enzymatic activity; 
 wherein the sample is suspected of comprising cells of interest that comprise an intracellular enzyme having the measurable enzymatic activity; 
 wherein treating the liquid sample comprises treating the sample with a reagent that inactivates the measurable enzymatic activity in the extracellular medium, but does not inactivate the measurable enzymatic activity in the cells of interest; 
   establishing a culture of the cells of interest remaining after the sample is treated;   removing a first portion of the established culture;   lysing cells of interest in the first portion to form a first lysate; and   measuring the measurable enzymatic activity of the first lysate.   
     
     
         11 . The method of  claim 10 , further comprising:
 removing a second portion of the established culture of the cells of interest;   lysing cells of interest in the second portion to form a second lysate; and   measuring the measurable enzymatic activity of the second lysate.   
     
     
         12 . The method of  claim 11 , wherein the second portion is removed at a point in time after the first portion is removed. 
     
     
         13 . The method of  claim 12 , further comprising comparing the measurable enzymatic activity of the first lysate with the measurable enzymatic activity of the second lysate. 
     
     
         14 . A method comprising:
 treating a liquid sample comprising an extracellular medium;
 wherein the extracellular medium contains an extracellular enzyme having a measurable enzymatic activity; 
 wherein the sample is suspected of comprising cells of interest that comprise an intracellular enzyme having the measurable enzymatic activity; 
 wherein treating the liquid sample comprises treating the sample with a reagent that inactivates the measurable enzymatic activity in the extracellular medium, but does not inactivate the measurable enzymatic activity in the cells of interest; 
   after treating the liquid sample, establishing a culture of the cells of interest from the treated liquid sample;   removing a first subsample and a second subsample from the culture;   treating the first subsample with an antimicrobial agent;   leaving the second subsample untreated with the antimicrobial agent; and   comparing cell growth in the first subsample to cell growth in the second subsample.   
     
     
         15 . The method of  claim 14 , further comprising:
 removing a third subsample from the culture;   treating the first subsample with a first antimicrobial agent;   treating the third subsample with a second antimicrobial agent; and   comparing cell growth in the third subsample to cell growth in the second subsample.