View More View Less
  • 1 Semmelweis Egyetem, Általános Orvostudományi Kar Genetikai, Sejt- és Immunbiológiai Intézet Budapest Nagyvárad tér 4. 1089
Open access

A kórokozók közvetlen kimutatásán alapuló mikrobiológiai diagnosztika idő- és munkaigényes folyamat. Nem csoda tehát, hogy megnőtt az igény a konvencionális módszereknél gyorsabb, nagy érzékenységű diagnosztikai eljárások kifejlesztése és bevezetése iránt, mint amilyenek a nukleinsav-kimutatáson alapuló módszerek. A molekuláris biológiai módszerek felgyorsítják a diagnosztikát, javítják a specificitást és lehetőséget nyújtanak a nehezen tenyészthető patogének azonosítására is. Az eredmények értékelésekor azonban figyelembe kell venni, hogy a kórokozó-specifikus nukleinsav kimutatása nem egyenértékű a szaporodni képes kórokozók jelenlétével! Az áramlási citometria legnagyobb előnye, a nukleinsav-detektáláson alapuló technikákkal szemben, a mikroorganizmusok egyedi, sejtszintű kimutatása. Gyorsasága révén kiemelt jelentőségre tehet szert a lassan növő mikrobák, a Mycobacterium törzsek és a gombák kimutatásában. Mivel a megfelelő minta-előkészítés megválasztásával alkalmas a patogének pontos azonosítására, így különösen jól hasznosítható a kevert populációk okozta fertőzések diagnosztikájában. Nem utolsósorban lehetővé teszi a fertőzött szervezet immunrendszerének monitorozását és az antimikrobiális kezelés nyomon követését is. Orv. Hetil., 2013, 154, 1207–1218.

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • De Araujo Souza, P. S., Villa, L. L.: Genetic susceptibility to infection with human papillomavirus and development of cervical cancer in women in Brazil. Mutat. Res., 2003, 544, 375–383.

    Villa L. L. , 'Genetic susceptibility to infection with human papillomavirus and development of cervical cancer in women in Brazil ' (2003 ) 544 Mutat. Res. : 375 -383.

    • Search Google Scholar
  • Hofstraat, J. W., van Zeijl, W. J. M., de Vreeze, M. E. J., et al.: Phytoplankton monitoring by flow cytometry. J. Plankton Res., 1994, 16, 1197–1224.

    Vreeze M. E. J. , 'Phytoplankton monitoring by flow cytometry ' (1994 ) 16 J. Plankton Res. : 1197 -1224.

    • Search Google Scholar
  • Rychlik, I., Cardova, L., Sevcik, M., et al.: Flow cytometry characterisation of Salmonella typhimurium mutants defective in proton translocating proteins and stationary-phase growth phenotype. J. Microbiol. Methods, 2000, 42, 255–263.

    Sevcik M. , 'Flow cytometry characterisation of Salmonella typhimurium mutants defective in proton translocating proteins and stationary-phase growth phenotype ' (2000 ) 42 J. Microbiol. Methods : 255 -263.

    • Search Google Scholar
  • Sizemore, R. K., Caldwell, J. J., Kendrick, A. S.: Alternate Gram staining technique using a fluorescent lectin. Appl. Environ. Microbiol., 1990, 56, 2245–2247.

    Kendrick A. S. , 'Alternate Gram staining technique using a fluorescent lectin ' (1990 ) 56 Appl. Environ. Microbiol. : 2245 -2247.

    • Search Google Scholar
  • Allman, R., Manchee, R., Lloyd, D.: Flow cytometric analysis of heterogeneous bacterial populations. In: Flow cytometry in microbiology. Springer-Verlag, London, 1993, 27–47.

    Lloyd D. , '', in Flow cytometry in microbiology , (1993 ) -.

  • Shapiro, H. M.: Practical flow cytometry. A. R. Liss, Inc., New York, 1995, 367–425.

    Shapiro H. M. , '', in Practical flow cytometry , (1995 ) -.

  • Mason, D. J., Shanmuganathan, S., Mortimer, F. C., et al.: A fluorescent Gram stain for flow cytometry and epifluorescence microscopy. Appl. Environ. Microbiol., 1998, 64, 2681–2685.

    Mortimer F. C. , 'A fluorescent Gram stain for flow cytometry and epifluorescence microscopy ' (1998 ) 64 Appl. Environ. Microbiol. : 2681 -2685.

    • Search Google Scholar
  • McClelland, R. G., Pinder, A. C.: Detection of low levels of specific Salmonella species by fluorescent antibodies and flow cytometry. J. Appl. Bacteriol., 1994, 77, 440–447.

    Pinder A. C. , 'Detection of low levels of specific Salmonella species by fluorescent antibodies and flow cytometry ' (1994 ) 77 J. Appl. Bacteriol. : 440 -447.

    • Search Google Scholar
  • Ozanne, V., Ortalo-Magne, A., Vercellone, A., et al.: Cytometric detection of mycobacterial surface antigens: exposure of mannosyl epitopes and of the arabinan segment of arabinomannans. J. Bacteriol., 1996, 178, 7254–7259.

    Vercellone A. , 'Cytometric detection of mycobacterial surface antigens: exposure of mannosyl epitopes and of the arabinan segment of arabinomannans ' (1996 ) 178 J. Bacteriol. : 7254 -7259.

    • Search Google Scholar
  • Fernandez-Prada, C. M., Zelazowska, E. B., Bhattacharjee, A. K., et al.: Identification of smooth and rough forms in cultures of Brucella melitensis strains by flow cytometry. J. Immunol. Methods, 2006, 315, 162–170.

    Bhattacharjee A. K. , 'Identification of smooth and rough forms in cultures of Brucella melitensis strains by flow cytometry ' (2006 ) 315 J. Immunol. Methods : 162 -170.

    • Search Google Scholar
  • Stender, H., Lund, K., Petersen, K. H., et al.: Fluorescence in situ hybridization assay using peptide nucleic acid probes for differentiation between tuberculous and nontuberculous mycobacterium species in smears of mycobacterium cultures. J. Clin. Microbiol., 1999, 37, 2760–2765.

    Petersen K. H. , 'Fluorescence in situ hybridization assay using peptide nucleic acid probes for differentiation between tuberculous and nontuberculous mycobacterium species in smears of mycobacterium cultures ' (1999 ) 37 J. Clin. Microbiol. : 2760 -2765.

    • Search Google Scholar
  • Han, Y., Kanbe, T., Cherniak, R., et al.: Biochemical characterization of Candida albicans epitopes that can elicit protective and nonprotective antibodies. Infect. Immun., 1997, 65, 4100–4107.

    Cherniak R. , 'Biochemical characterization of Candida albicans epitopes that can elicit protective and nonprotective antibodies ' (1997 ) 65 Infect. Immun. : 4100 -4107.

    • Search Google Scholar
  • Mercure, S., Sénéchal, S., Auger, P., et al.: Candida albicans serotype analysis by flow cytometry. J. Clin. Microbiol., 1996, 34, 2106–2112.

    Auger P. , 'Candida albicans serotype analysis by flow cytometry ' (1996 ) 34 J. Clin. Microbiol. : 2106 -2112.

    • Search Google Scholar
  • Piérard, G. E., Arrese, J. E., De Doncker, P., et al.: Present and potential diagnostic techniques in onychomycosis. J. Am. Acad. Dermatol., 1996, 34 (2 Pt 1), 273–277.

    Doncker P. , 'Present and potential diagnostic techniques in onychomycosis ' (1996 ) 34 J. Am. Acad. Dermatol. : 273 -277.

    • Search Google Scholar
  • Di Giorgio, C., Ridoux, O., Delmas, F., et al.: Flow cytometric detection of Leishmania parasites in human monocyte-derived macrophages: application to antileishmanial-drug testing. Antimicrob. Agents Chemother., 2000, 44, 3074–3078.

    Delmas F. , 'Flow cytometric detection of Leishmania parasites in human monocyte-derived macrophages: application to antileishmanial-drug testing ' (2000 ) 44 Antimicrob. Agents Chemother. : 3074 -3078.

    • Search Google Scholar
  • Flores, B. M., Garcia, C. A., Stamm, W. E., et al.: Differentiation of Naegleria fowleri from Acanthamoeba species by using monoclonal antibodies and flow cytometry. J. Clin. Microbiol., 1990, 28, 1999–2005.

    Stamm W. E. , 'Differentiation of Naegleria fowleri from Acanthamoeba species by using monoclonal antibodies and flow cytometry ' (1990 ) 28 J. Clin. Microbiol. : 1999 -2005.

    • Search Google Scholar
  • Uehlinger, F. D., Barkema, H. W., O’Handley, R. M., et al.: Comparison of flow cytometry and immunofluorescence microscopy for the detection of Giardia duodenalis in bovine fecal samples. J. Vet. Diagn. Invest., 2008, 20, 178–185.

    O’Handley R. M. , 'Comparison of flow cytometry and immunofluorescence microscopy for the detection of Giardia duodenalis in bovine fecal samples ' (2008 ) 20 J. Vet. Diagn. Invest. : 178 -185.

    • Search Google Scholar
  • Iannelli, D., D’Apice, L., Cottone, C., et al.: Simultaneous detection of cucumber mosaic virus, tomato mosaic virus and potato virus Y by flow cytometry. J. Virol. Methods, 1997, 69, 137–145.

    Cottone C. , 'Simultaneous detection of cucumber mosaic virus, tomato mosaic virus and potato virus Y by flow cytometry ' (1997 ) 69 J. Virol. Methods : 137 -145.

    • Search Google Scholar
  • Elmendorf, S., McSharry, J., Laffin, J., et al.: Detection of an early cytomegalovirus antigen with two-color quantitative flow cytometry. Cytometry, 1988, 9, 254–260.

    Laffin J. , 'Detection of an early cytomegalovirus antigen with two-color quantitative flow cytometry ' (1988 ) 9 Cytometry : 254 -260.

    • Search Google Scholar
  • Papsidero, L. D., Dittmer, R. P., Vaickus, L., et al.: Monoclonal antibodies and chemiluminescence immunoassay for detection of the surface protein of human T-cell lymphotropic virus. J. Clin. Microbiol., 1992, 30, 351–358.

    Vaickus L. , 'Monoclonal antibodies and chemiluminescence immunoassay for detection of the surface protein of human T-cell lymphotropic virus ' (1992 ) 30 J. Clin. Microbiol. : 351 -358.

    • Search Google Scholar
  • McSharry, J. J., Costantino, R., McSharry, M. B., et al.: Rapid detection of herpes simplex virus in clinical samples by flow cytometry after amplification in tissue culture. J. Clin. Microbiol., 1990, 28, 1864–1866.

    McSharry M. B. , 'Rapid detection of herpes simplex virus in clinical samples by flow cytometry after amplification in tissue culture ' (1990 ) 28 J. Clin. Microbiol. : 1864 -1866.

    • Search Google Scholar
  • Just, T., Burgwald, H., Broe, M. K.: Flow cytometric detection of EBV (EBER snRNA) using peptide nucleic acid probes. J. Virol. Methods, 1998, 73, 163–174.

    Broe M. K. , 'Flow cytometric detection of EBV (EBER snRNA) using peptide nucleic acid probes ' (1998 ) 73 J. Virol. Methods : 163 -174.

    • Search Google Scholar
  • Snoeck, R., Schols, D., Sadzot-Delvaux, C., et al.: Flow cytometric method for the detection of gpI antigens of varicella zoster virus and evaluation of anti-VZV agents. J. Virol. Methods, 1992, 38, 243–254.

    Sadzot-Delvaux C. , 'Flow cytometric method for the detection of gpI antigens of varicella zoster virus and evaluation of anti-VZV agents ' (1992 ) 38 J. Virol. Methods : 243 -254.

    • Search Google Scholar
  • Chemin, I., Vermot-Desroches, C., Baginski, I., et al.: Selective detection of human hepatitis B virus surface and core antigens in peripheral blood mononuclear cell subsets by flow cytometry. J. Viral. Hepat., 1994, 1, 39–44.

    Baginski I. , 'Selective detection of human hepatitis B virus surface and core antigens in peripheral blood mononuclear cell subsets by flow cytometry ' (1994 ) 1 J. Viral. Hepat. : 39 -44.

    • Search Google Scholar
  • El-Awady, M. K., Tabll, A. A., Redwan, el-R. M., et al.: Flow cytometric detection of hepatitis C virus antigens in infected peripheral blood leukocytes: binding and entry. World J. Gastroenterol., 2005, 11, 5203–5208.

    Redwan el-R. M. , 'Flow cytometric detection of hepatitis C virus antigens in infected peripheral blood leukocytes: binding and entry ' (2005 ) 11 World J. Gastroenterol. : 5203 -5208.

    • Search Google Scholar
  • Bordignon, J., Pires Ferreira, S. C., Medeiros Caporale, G. M., et al.: Flow cytometry assay for intracellular rabies virus detection. J. Virol. Methods, 2002, 105, 181–186.

    Medeiros Caporale G. M. , 'Flow cytometry assay for intracellular rabies virus detection ' (2002 ) 105 J. Virol. Methods : 181 -186.

    • Search Google Scholar
  • Honda, J., Okubo, Y., Imamura, Y., et al.: Flow cytometric detection of cytomegalovirus antigen in peripheral blood cells after bone marrow transplantation. Br. J. Haematol., 1997, 99, 415–417.

    Imamura Y. , 'Flow cytometric detection of cytomegalovirus antigen in peripheral blood cells after bone marrow transplantation ' (1997 ) 99 Br. J. Haematol. : 415 -417.

    • Search Google Scholar
  • Re, M. C., Furlini, G., Gibellini, D., et al.: Quantification of human immunodeficiency virus type 1-infected mononuclear cells in peripheral blood of seropositive subjects by newly developed flow cytometry analysis of the product of an in situ PCR assay. J. Clin. Microbiol., 1994, 32, 2152–2157.

    Gibellini D. , 'Quantification of human immunodeficiency virus type 1-infected mononuclear cells in peripheral blood of seropositive subjects by newly developed flow cytometry analysis of the product of an in situ PCR assay ' (1994 ) 32 J. Clin. Microbiol. : 2152 -2157.

    • Search Google Scholar
  • Lichtner, M., Marañón, C., Azocar, O., et al.: HIV type 1-infected dendritic cells induce apoptotic death in infected and uninfected primary CD4 T lymphocytes. AIDS Res. Hum. Retroviruses, 2004, 20, 175–182.

    Azocar O. , 'HIV type 1-infected dendritic cells induce apoptotic death in infected and uninfected primary CD4 T lymphocytes ' (2004 ) 20 AIDS Res. Hum. Retroviruses : 175 -182.

    • Search Google Scholar
  • Hunt, H. D., Lupiani, B., Miller, M. M., et al.: Marek’s disease virus down-regulates surface expression of MHC (B complex) class I (BF) glycoproteins during active but not latent infection of chicken cells. Virology, 2001, 282, 198–205.

    Miller M. M. , 'Marek’s disease virus down-regulates surface expression of MHC (B complex) class I (BF) glycoproteins during active but not latent infection of chicken cells ' (2001 ) 282 Virology : 198 -205.

    • Search Google Scholar
  • Inghirami, G., Nakamura, M., Balow, J. E., et al.: Model for studying virus attachment: identification and quantitation of Epstein-Barr virus-binding cells by using biotinylated virus in flow cytometry. J. Virol., 1988, 62, 2453–2463.

    Balow J. E. , 'Model for studying virus attachment: identification and quantitation of Epstein-Barr virus-binding cells by using biotinylated virus in flow cytometry ' (1988 ) 62 J. Virol. : 2453 -2463.

    • Search Google Scholar
  • Kaprelyants, A. S., Mukamolova, G. V., Davey, H. M., et al.: Quantitative analysis of the physiological heterogeneity within starved cultures of Micrococcus luteus by flow cytometry and cell sorting. Appl. Environ. Microbiol., 1996, 62, 1311–1316.

    Davey H. M. , 'Quantitative analysis of the physiological heterogeneity within starved cultures of Micrococcus luteus by flow cytometry and cell sorting ' (1996 ) 62 Appl. Environ. Microbiol. : 1311 -1316.

    • Search Google Scholar
  • Joux, F., Lebaron, P.: Use of fluorescent probes to assess physiological functions of bacteria at single-cell level. Microbes Infect., 2000, 2, 1523–1535.

    Lebaron P. , 'Use of fluorescent probes to assess physiological functions of bacteria at single-cell level ' (2000 ) 2 Microbes Infect. : 1523 -1535.

    • Search Google Scholar
  • Quirós, C., Herrero, M., García, L. A., et al.: Application of flow cytometry to segregated kinetic modeling based on the physiological states of microorganisms. Appl. Environ. Microbiol., 2007, 73, 3993–4000.

    García L. A. , 'Application of flow cytometry to segregated kinetic modeling based on the physiological states of microorganisms ' (2007 ) 73 Appl. Environ. Microbiol. : 3993 -4000.

    • Search Google Scholar
  • Del Giorgio, P. A., Prairie, Y. T., Bird, D. F.: Coupling between rates of bacterial production and the abundance of metabolically active bacteria in lakes, enumerated using CTC reduction and flow cytometry. Microb. Ecol., 1997, 34, 144–154.

    Bird D. F. , 'Coupling between rates of bacterial production and the abundance of metabolically active bacteria in lakes, enumerated using CTC reduction and flow cytometry ' (1997 ) 34 Microb. Ecol. : 144 -154.

    • Search Google Scholar
  • Nedoma, J., Vrba, J.: Specific activity of cell-surface acid phosphatase in different bacterioplankton morphotypes in an acidified mountain lake. Environ. Microbiol., 2006, 8, 1271–1279.

    Vrba J. , 'Specific activity of cell-surface acid phosphatase in different bacterioplankton morphotypes in an acidified mountain lake ' (2006 ) 8 Environ. Microbiol. : 1271 -1279.

    • Search Google Scholar
  • Huang, Z., You, W., Haugland, R. P., et al.: A novel fluorogenic substrate for detecting alkaline phosphatase activity in situ. J. Histochem. Cytochem., 1993, 41, 313–317.

    Haugland R. P. , 'A novel fluorogenic substrate for detecting alkaline phosphatase activity in situ ' (1993 ) 41 J. Histochem. Cytochem. : 313 -317.

    • Search Google Scholar
  • Duhamel, S., Gregori, G., Van Wambeke, F., et al.: A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry. J. Microbiol. Methods, 2008, 75, 269–278.

    Wambeke F. , 'A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry ' (2008 ) 75 J. Microbiol. Methods : 269 -278.

    • Search Google Scholar
  • Berney, M., Weilenmann, H. U., Egli, T.: Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS). Microbiology, 2006, 152, 1719–1729.

    Egli T. , 'Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS) ' (2006 ) 152 Microbiology : 1719 -1729.

    • Search Google Scholar
  • Ozanich, R. M. Jr., Bruckner-Lea, C. J., Warner, M. G., et al.: Rapid multiplexed flow cytometric assay for botulinum neurotoxin detection using an automated fluidic microbead-trapping flow cell for enhanced sensitivity. Anal. Chem., 2009, 81, 5783–5793.

    Warner M. G. , 'Rapid multiplexed flow cytometric assay for botulinum neurotoxin detection using an automated fluidic microbead-trapping flow cell for enhanced sensitivity ' (2009 ) 81 Anal. Chem. : 5783 -5793.

    • Search Google Scholar
  • Etienne, K. A., Kano, R., Balajee, S. A.: Development and validation of a microsphere-based Luminex assay for rapid identification of clinically relevant aspergilli. J. Clin. Microbiol., 2009, 47, 1096–1100.

    Balajee S. A. , 'Development and validation of a microsphere-based Luminex assay for rapid identification of clinically relevant aspergilli ' (2009 ) 47 J. Clin. Microbiol. : 1096 -1100.

    • Search Google Scholar
  • Martinez, O. V., Gratzner, H. G., Malinin, T. I., et al.: The effect of some beta-lactam antibiotics on Escherichia coli studied by flow cytometry. Cytometry, 1982, 3, 129–133.

    Malinin T. I. , 'The effect of some beta-lactam antibiotics on Escherichia coli studied by flow cytometry ' (1982 ) 3 Cytometry : 129 -133.

    • Search Google Scholar
  • Pina-Vaz, C., Costa-de-Oliveira, S., Rodrigues, A. G.: Safe susceptibility testing of Mycobacterium tuberculosis by flow cytometry with the fluorescent nucleic acid stain SYTO 16. J. Med. Microbiol., 2005, 54, 77–81.

    Rodrigues A. G. , 'Safe susceptibility testing of Mycobacterium tuberculosis by flow cytometry with the fluorescent nucleic acid stain SYTO 16 ' (2005 ) 54 J. Med. Microbiol. : 77 -81.

    • Search Google Scholar
  • Kelly, D. J., Salata, K. F., Strickman, D., et al.: Rickettsia tsutsugamushi infection in cell culture: antibiotic susceptibility determined by flow cytometry. Am. J. Trop. Med. Hyg., 1995, 53, 602–606.

    Strickman D. , 'Rickettsia tsutsugamushi infection in cell culture: antibiotic susceptibility determined by flow cytometry ' (1995 ) 53 Am. J. Trop. Med. Hyg. : 602 -606.

    • Search Google Scholar
  • Dessus-Babus, S., Belloc, F., Bébéar, C. M., et al.: Antibiotic susceptibility testing for Chlamydia trachomatis using flow cytometry. Cytometry, 1998, 31, 37–44.

    Bébéar C. M. , 'Antibiotic susceptibility testing for Chlamydia trachomatis using flow cytometry ' (1998 ) 31 Cytometry : 37 -44.

    • Search Google Scholar
  • Balkis, M. M., Leidich, S. D., Mukherjee, P. K., et al.: Mechanisms of fungal resistance: an overview. Drugs, 2002, 62, 1025–1040.

    Mukherjee P. K. , 'Mechanisms of fungal resistance: an overview ' (2002 ) 62 Drugs : 1025 -1040.

    • Search Google Scholar
  • Di Giorgio, C., Ridoux, O., Delmas, F., et al.: Flow cytometric detection of Leishmania parasites in human monocyte-derived macrophages: application to antileishmanial-drug testing. Antimicrob. Agents Chemother, 2000, 44, 3074–3078.

    Delmas F. , 'Flow cytometric detection of Leishmania parasites in human monocyte-derived macrophages: application to antileishmanial-drug testing ' (2000 ) 44 Antimicrob. Agents Chemother : 3074 -3078.

    • Search Google Scholar
  • Katsuragi, T., Tani, Y.: Screening for microorganisms with specific characteristics by flow cytometry and single-cell sorting. J. Biosci. Bioeng., 2000, 89, 217–222.

    Tani Y. , 'Screening for microorganisms with specific characteristics by flow cytometry and single-cell sorting ' (2000 ) 89 J. Biosci. Bioeng. : 217 -222.

    • Search Google Scholar
  • Harari, A., Zimmerli, S. C., Pantaleo, G.: Cytomegalovirus (CMV)-specific cellular immune responses. Hum. Immunol., 2004, 65, 500–506.

    Pantaleo G. , 'Cytomegalovirus (CMV)-specific cellular immune responses ' (2004 ) 65 Hum. Immunol. : 500 -506.

    • Search Google Scholar