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• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/06—Quantitative determination
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
  • G01N33/56933—Mycoplasma

Definitions

• the present invention describes a method for the direct detection of Mycobacterium tuberculosis (hereafter, referred to as M.TB.) infection, inside phagocytes collected from peripheral blood, sputum, bronchoalveolar lavage, serositis exudates and, in general, from every biological fluid or tissue sample accrued from the human body.
• M.TB. Mycobacterium tuberculosis
• the said detection involves immunofluoresence in combination with flow cytometry.
• Tuberculosis is a modern plague of civilization. Globally, a total of 13.7 million prevalent TB cases were recorded in 2007, with an estimated 1.37 million of incident TB cases in 2007 coinfected with HIV. This corresponds to a prevalence of 206 cases per 100,000 that resulted in 1.756 million deaths (including 456,000 TB patients coinfected with HIV) worldwide. 500,000 cases of multidrug-resistant TB (MDRTB) defined as infection with M. tuberculosis strains resistant to at least two of the most important first-line drugs, rifampin and isoniazid) occurred in 2007 [1].
• MDRTB multidrug-resistant TB
• XDR-TB drug-resistant TB
• MDRTB strains additionally resistant to a fluoroquinolone and an injectable agent such as kanamy- cin, amikacin, viomycin, or capreomycin
• kanamy- cin a fluoroquinolone
• an injectable agent such as kanamy- cin, amikacin, viomycin, or capreomycin
• the World Health Organization has estimated that one-third of the total world population is latently infected with M. tuberculosis (hereafter, referred as LTB) and that 5%-10% of infected individuals will develop active TB disease during their life time. However, the risk of developing active disease is 5%-15% every year and lifetime risk is 50% in HIV coinfected individuals. Most of the active disease cases in low TB incidence countries arise from this pool of latently infected individuals [1 , 2].
• LTBI individuals The number of LTBI individuals is anticipated to rise, especially in developed countries, due to the increased number of medically immunocompromised patients, such as organ transplant recipients and cancer patients, as well as patients on treatment with biological agents (anti-TNF-a agents, etc.).
• a large number of refu- gees from countries with high inidences of TB presumably will emigrate due to political and economic problems.
• M.Tb. Primary infection with M.Tb. results in a disease state in only 10% of infected individuals. The rest of the infected population develops an induced immunological re- sponse, thereby averting further spread of M.Tb. which is confined to cellular complexes called tubercles. M.TB. cannot multiply in tubercles and as a result remains latent. The host, therefore, remains healthy although the pathogen could potentially revert to its virulent state, especially when the host is under reduced immunosur- veilance, i.e. in the case of HIV infection.
• the currently known tests for diagnosis of LTBI are based exclusively on immunological tests assesing host-specific cellular immunity to MTBC antigens.
• IFN-y immunity-based interferon-gamma
• tuberculosis-specific antigens are the main tests for LTBI [3].
• IGRA assays include QuantiFERON-TB Gold, QuantiFERON-TB Gold In- Tube (QFT-G-IT), and T-SPOT.TB
• QFT-G-IT QuantiFERON-TB Gold In- Tube
• T-SPOT.TB T-SPOT.TB
• the pooled sensitivity of IGRAs was 70% - 90% and the pooled specificity was 93% - 99%.
• the pooled sensitivity for the diagnosis of active TB was 80% - and 48%, respectively, for QFT-G-IT.
• the respective sensitivity for T-SPOT was 81 % and 88%.
• the pooled specificity was 79% and 82% for QFT-G-IT, and 59% and 82% for T-SPOT.TB, respectively [4].
• the conventional microbiology test i.e. direct smear and culture test
• the conventional microbiology test also lacks sensitivity for the diagnosis of pulmonary TB, with an estimated 30% of active pulmonary TB cases appearing as sputum smear and culture negative.
• culture-restricted results are delayed, emerging after a long time(aprox. after fifteen days).
• NAAT Nucleic Acid Amplification Test
• the present invention solves the above described problems and for the first time provides a direct, sensitive and specific method for M.TB. detection, not only in LTB cases, but also in those cases accompanied by severe forms of tuberculosis.
• the primary route of infection from M.TB. involves the lungs.
• Inhaled droplet nuclei avoid bronnhal ripf nsps HUP tr> thpir small c anH thta terminal al_ veoli where they are engulfed by phagocytic immune cells (macrophages and dendritic cells).
• tuberculosis can also infect nonphagocytic cells in the alveolar space, including alveolar endothelial, and type 1 and type 2 epithelial cells (pneumocytes) (6).
• a large number of microbes is transfered via macrophages to draining lymph nodes.
• region of difference 1 region of difference 1
• M. tuberculosis a genomic region that is present in all virulent M. tuberculosis and M. bovis strains but absent in the vaccine strain M. bovis BCG (7-9).
• RD1 region of difference 1
• the virulence of M. tuberculosis stems from RD1 products and especially the ESAT-6 and CFP-10 proteins.
• Region RD1 is an important research field for new vaccines, as it offers more effective and promising venues than the BCG vaccine as well as new therapies.
• ESAT-6 causes cy- tolysis of type 1 and type 2 pneumocytes and that ESAT-6 induced lysis contributes to bacilli dissemination to alveolar wall (6).
• ESAT-6 is linked to metabolically active M.TB.
• its intracellular expression should be a usefull biomarker not only for active tuberculosis but also for LTB. Therefore, we considered that this intracellular expression of ESAT-6 should have a prognostic value as regards either LTB or tuberculosis diagnosis, because it indirectly identifies, via ESAT-6, live and virulent bacilli, especially in cases that sensitivity is too low.
• ESAT-6 has not been 'targeted' by anyone as an activity marker of M.TB.
• the inventors consider vital the search for the presence of ESAT-6 in the interior of cells targeted by M.TB. bacilli, such as leukocytes, bronchoalveolar lavage macrophages, cells from cerebrospinal fluid, pleural effusions, pericardial effusions, synovial fluids, sperm, induced sputum, vaginal secretions and in general every biological fluid sample collected from the human body and cell suspensions from serositis effusions, mucosal eluants or solid tissues.
• bacilli such as leukocytes, bronchoalveolar lavage macrophages, cells from cerebrospinal fluid, pleural effusions, pericardial effusions, synovial fluids, sperm, induced sputum, vaginal secretions and in general every biological fluid sample collected from the human body and cell suspensions from serositis effusions, mucosal eluants or solid tissues.
• the present invention describes a method of indirect detection of activeM.TB. inside cells via the cytoplasmic detection of ESAT-6, using either indirect or direct immunofluorescence techniques, followed by visualization and evaluation of the resut by flow cytometry
• the current invention describes a method for intracellular detection using specific antibodies against RD1- M.TB. genome products in human cells removed from the human organism.
• the intracellular detection method set forth in this invention combines the immunofluorescence technique with flow cytometry.
• any fluorochrome can be used for fluorescence.
• the said fluorochromes can be selected from the following: Fluorecein- 5- isothiocyanate (FITC), aminomethylcoumarin Acetate (AMCA 350), 6,8-difluoro-7- hydroxycoumarin derivative (Marina Blue), Cascade Blue, Alexa fluor 405, 6,8- difluoro-7-hydroxycoumarin derivative (Pacific Blue), Alexa Fluor 430, Cascade Yellow, Alexa Fluor 488, phycoerythrin (PE), phycoerythrin Texas Red (PE-Texas Red), phycoerythrin-cyanin 5 (PE-Cy5), peridinin chlorophyll protein (PerCP), peridinin chlorophyll protein -cyanin 5.5 (PerCP-Cy5.5), phycoerythrin-cyanin 7( PE-
• cells to be assayed as in the current invention should preferably come from isolated pleural effusions, pericardial effusions, synovial fluids, sperm, induced sputum, vaginal secretions, in general every biological fluid sample collected from the human body and cell suspensions from serositis effusions, mucosal eluants or solid tissues, which previously had been removed from the human organism.
• the antibody used in the method of the present invention is a specific type of anti-ESAT-6.
• an additional stage of fluorophore-conjugated antibodies that recognize human antigen epitopes for infected cell determination can also be integrated into the whole procedure.
• the present invention also describes a kit for its implementation.
• the said kit includes either an anti-ESAT-6 antibody or another antibody against other RD1 prod- ucts of the M.TB. genome, together with additional reagents for the intracellular detection of the M.TB. bacillus in human cells that have already been removed from human organisms.lt is advantageous that the above described kit comprises a combination of fluorophore-conjugated antibodies against human antigen epitopes. ⁇
• the present invention discloses an automated flow cytometer, in which the whole procedure is performed automatically (smart flow cytometry).
• the said automated flow cytometer incorporates the kit for the direct detection of RD1 products in parallel with the evaluation of any other phenotypical parameter (sub- populations of CD4, CD8 cells), and in combination with the detection and characrerization of viruses.
• the apparatus described by the present invention designates RD1 products such as ESAT-6, CFP-10, TB7.7, Ag85 etc, in cells collected from all biological fluids that have already been removed from the human organism.
• the apparatus described in the present invention can perform leuko- cyte functional assays and phenotypical analysis, i.e. it can assess the CD4/CD8 index in HIV patients from cells that have already been removed from the human organism.
• the method of the present invention comprises the following stages: cell fixation, direct or indirect intracellular immunofluorescence, visualization and evaluation of the result with flow cytometry.
• the method of the present invention detects active tuberculosis with the identification of a sequence of the ESAT-6 protein. It is also feasible to target other RD1 products with a view to obtaining the same result
• the method of the present invention for immufluorescence employs any anti- antibody conjugated with suitable fluorescence and can use any fluorophore, comprising any of the following currently known: Fluorecein-5- isothiocyanate (FITC), aminomethylcoumarin Acetate (AMCA 350), 6,8-difluoro-7-hydroxycoumarin derivative (Marina Blue), Cascade Blue, Alexa fluor 405, 6,8-difluoro-7-hydroxycoumarin derivative (Pacific Blue), Alexa Fluor 430, Cascade Yellow, Alexa Fluor 488, phyco- erythrin (PE), phycoerythrin Texas Red (PE-Texas Red), phycoerythrin-cyanin 5 (PE-Cy5) , peridinin chlorophyll protein (PerCP), peridinin chlorophyll protein-cyanin 5.5 (PerCP-Cy5.5), phycoerythrin-cyanin 7( PE-Cy7), Rhodamine TR, allophycocy
• the method of the present invention can additionally include an extracellular immunofluorescence procedure so that the cell's phenotype containing the bacilli can be identified.
• kits i.e. a product in a special package that includes reagents and necessary instructions for the assay procedure.
• the above kit comprises one or more antibodies against the ESAT-6 protein since it is our intention to detect it. If these antibodies are conjugated, the anti-antibody is not necessary; in case the antibodies are not conjugated, the kit will further comprise a second antibody (anti-antibody) conjugated to a fluorophore or biotin.
• An example of the implementation of the current invention is the following: we choose to look for the presence of ESAT-6 in the peripheral blood of a patient with active disease.
• a normal donor who is negative for any manifestation of disease in all clinical assays that assess tuberculosis is used as a negative control. 1 Fixation of blood cells
• PFA Paraformaldehyde
• indirect immnofluorescence is used, because it is is less costly than having all the antibodies directly conjugated.
• a fraction of the cells is spun down, resuspended, and incubated for 30 min in 100 ⁇ Phosphate Buffered Saline (PBS)-0.1% saponin (medium A). Saponin creates pores at the cellular membrane making intracellular access feasible. Therefore, an antibody namely, one intracellulary targeting the ESAT-6 antigen epitope (11G4) can be linked to its target epitope.
• PBS Phosphate Buffered Saline
• saponin creates pores at the cellular membrane making intracellular access feasible. Therefore, an antibody namely, one intracellulary targeting the ESAT-6 antigen epitope (11G4) can be linked to its target epitope.
• the cells are spun down again, the supernatant is discarded, the pellet resuspended in 50 ⁇ of medium A and incubated with titered amounts of,. the antibody against the ESAT-6 epitope, such as for example, 11G4 which targets the EQQWNFAGIEAAA epitope.
• the cells are then washed with 2ml PBS containing 0.1% saponin and 2% Fetal Calf Serum (FCS) (wash buffer-WB) and the supernatant is discarded.
• FCS Fetal Calf Serum
• the cell pellet is resuspended and a new incubation with a tittered amount of a polyclonal fluorophore-conjugated antibody against immunoglobulins from the animal from which the first antibody (fluorophore conjugated anti-antibody) was developed, follows.
• fluorophore can be used.
• the following are the most commonly known fluorophores; they are listed here indicatively and are not limited to: Fluorecein-5- isothiocyanate (FITC), aminomethylcoumarin Acetate (AMCA 350), 6,8-difluoro-7- hydroxycoumarin derivative (Marina Blue), Cascade Blue, Alexa fluor 405, 6,8- difluoro-7-hydroxycoumarin derivative (Pacific Blue), Alexa Fluor 430, Cascade Yellow, Alexa Fluor 488, phycoerythrin (PE), phycoerythrin Texas Red (PE-Texas RedV nhv rifirvth peridinin chlorophyll protein -cyanin 5.5 (PerCP-Cy5.5), phycoerythrin-cyanin 7( PE- Cy7), Rhodamine TR, allophycocyanin (APC), ALexa Fluor 647, allophycocyanin cyanin 7 (APC-Cy7), BD APC
• the cells are washed with 2 ml WB. The su- pernatant is discarded and the cell pellet is resuspended in 50 ⁇ of PBS. If it is necessary to perform a leukocyte study to determine presence of ESAT-6, the procedure goes on to next step. Alternatively, the procedure proceeds to the step where the cells are harvested.
• a classic immunofluorescence assay can be performed and the cells incubated with an antibody i.e. against granulocytes (anti CD13) which is directly conjugated with a different fluorochrome than that used for ESAT-6 assession. Following a 30 min incubation at 4°C, the samples are washed with 2ml PBS containing 2% FCS. The supernatant is discarded, the cells are resuspended in PBS and are ready for acquisition in a Flow Cytometer.
• the samples are acquired in a flow cytometry apparatus.
• the data analysis is per- formed using suitable software where we use region combinations based on scatter standards (size, complexity) and/or expression of leukocyte antigens.
• the analysis focuses on detection of the intracellular presence of the EQQWNFAGIEAAA sequence of ESAT-6, identified from the 11 G4 clone, in certain cell populations (as depicted in figure 1 D).
• the presence of the antigenic epitope has been analyzed in the interior of granulocytes based on their scatter characteristics.
• Figures 1A, 1 B, 1C show the intracellular detection of epitope EQQWNFA ⁇
• GIEAAA belonging to the ESAT-6 protein, in the granulocytes of an active tuberculosis patient with flow cytometry.
• the antibody is: anti-Mycobacterium tuberculosis, ESAT-6 Monoclonal Antibody, clone 11 G4 (Thermo SCIENTIFIC).
• This antibody is rpmmmpnrlpri mannfartnrorc nrt the li+ar- n-/ ⁇ > tri ⁇ ⁇ -, » ⁇ ⁇ « . ⁇ » U J (WB) use and NOT for immunofluorescence assays (Product Data Sheet).
• WB Western Data Sheet
• figure 1A a region has been drawn, which according to scatter characteristics corresponds to granulocytes and so we focus our analysis on this region.
• Figure 1 D shows cells in which the 1 G4 target sequence is detected. Specifically, as resulted from the analysis of the cytometer (FACScan, Becton Dickinson) data using the CellQuest analysis program, 0.13% of gated granulocytes express the ESAT-6 protein. In the upper left quadrant of figure 1 D, dots, assessing ESAT-6 positive granulocytes, are shown. We therefore, conclude the specificity of 1 1 G4 detec- tion.
• Figures 2A, 2B, 2C and 2D depict the same parameters, but the cells are from a healthy donor, free of tuberculosis.
• Figure 2A depicts a region including granulocytes, while figure 2B shows non specific staining with the sole presence of a second antibody.
• An immunophenotypic method of intracellular detection of M.TB. is dercibed.
• the main goal is the direct detection of M.TB. which causes either infection or latent infection.
• the presence of M.TB. infection is detected using an antibody against the intracellular presence of whatever product of the RD1 region, which substantiates the presence of metabolically active bacilli.
• the described method allows for ESAT-6 expression assessment in each and every cell, which results in higher accuracy.
• 30% of sputum smears are found to be negative for active and contagious tuberculosis, when any of the currently known diagnostic methods is applied.
• the immunophenotypic method of RD1 product intracellular detection is very accurate since the antibody used is monoclonal, which means that itr ecognizes only one epitope.
• a negative control is included, which substantiates the specificity.
• itdoes not cross-react with either humoral infectious intermediates or activation agents arising from previous stages of infection (memory).
• the retrospective analysis of results is also feasible, on demand.
• Centaae Of infected cells AjSliallv nrflni ie o fraction of the total cells.
• the percentage of granulocytes that stained positive for RD1 protein products, such as ESAT-6, represents the activity of infection.
• the method of intracellular detection of antigen is simple, fast, reproducible and has low cost.
• the result is delivered after 90 min- utes, following either blood draw or other material collection.
• Remote sample transport is safe, without special precautions, as is usualy the case with infectious material transportation, since sample virulence is neutralized with fixation, while the possibility of detecting already inactivated, via fixation, viruses such as HIV, andhepatitis also remains.
• the only necessary equipment for implementating the proposed method is a flow cytometry apparatus in combination with basic laboratory equipment (centrifuge, etc).
• the protocol does not demand specialized personel for its implementation since it is simple in its use.

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