polimerazuli jaWvuri reaqcia polimerazuli jaWvuri reaqcia realur droSirealur droSi

((Real-Time PCRReal-Time PCR))

m. axalaiam. axalaiatuberkulozisa da tuberkulozisa da filtvis daavadebaTa filtvis daavadebaTa erovnuli centri, erovnuli centri, erovnuli centraluri erovnuli centraluri mikobaqteriologiuri mikobaqteriologiuri laboratorialaboratoria

Georgia - Vanderbilt University, Nashville, Tennessee, USA, Georgia - Vanderbilt University, Nashville, Tennessee, USA, 2006-2008 CRDF project:2006-2008 CRDF project:"Monitoring Chemotherapy Regimen Efficacy by "Monitoring Chemotherapy Regimen Efficacy by Mycobacterium Tuberculosis mRNA Detection in Sputum Mycobacterium Tuberculosis mRNA Detection in Sputum Specimens”Specimens”

““mkurnalobis monitoringis efqturobis mkurnalobis monitoringis efqturobis Sefaseba Sefaseba M. Tuberculosis M. Tuberculosis rnm-is gamovleniT naxvelSi”rnm-is gamovleniT naxvelSi”

saqarTvelo-vanderbildis universiteti, neSvili, tenisi, aSS, saqarTvelo-vanderbildis universiteti, neSvili, tenisi, aSS, 2006-2006-

2008, 2008, CRDFCRDF

polimerazuli jaWvuri reaqciis polimerazuli jaWvuri reaqciis komponentebikomponentebi

dnm matricadnm matrica ori praimeri ori praimeri Termostabiluri dnm-polimeraza Termostabiluri dnm-polimeraza dezoqsinukleozidtrifosfatebidezoqsinukleozidtrifosfatebi Mg 2+ Mg 2+ ionebiionebi buferis xsnaribuferis xsnari

polimerazuli jaWvuri reaqciapolimerazuli jaWvuri reaqcia

denaturacia – dnm-s orjaWviani denaturacia – dnm-s orjaWviani molekulis molekulis gaxleCvagaxleCva

anilizacia – temperaturis swrafi anilizacia – temperaturis swrafi dawevis dawevis fonze praimerebis dakavSireba fonze praimerebis dakavSireba erTjaWvian erTjaWvian matricasTan matricasTan

elongacia – dnm-polimeraza axdens elongacia – dnm-polimeraza axdens matriculi jaWvis replikacias matriculi jaWvis replikacias

polimerazuli jaWvuri reaqciis polimerazuli jaWvuri reaqciis saxeobebisaxeobebi

ukutranskripciuli pjrukutranskripciuli pjr

asimetriuli pjr asimetriuli pjr

pjr dnm-s Tavisufali boloebis pjr dnm-s Tavisufali boloebis swrafi amplifikaciiTswrafi amplifikaciiT

raodenobrivi pjrraodenobrivi pjr

raodenobrivi pjr realur droSi raodenobrivi pjr realur droSi

raodenobrivi pjr realur droSiraodenobrivi pjr realur droSi

principi:principi: Cveulebrivi pjr Cveulebrivi pjr

magram:magram: dnm/rnm molekulebis dnm/rnm molekulebis

amplifikaciasTan erTad xdeba amplifikaciasTan erTad xdeba maTi maTi raodenobis gansazRvra raodenobis gansazRvra realur realur droSi droSi TiToeuli TiToeuli amplifikaciis amplifikaciis (ciklis) (ciklis) SemdegSemdeg

amplificirebuli dnm/rnm molekulebis amplificirebuli dnm/rnm molekulebis raodenobis gansazRvrisaTvis raodenobis gansazRvrisaTvis

gamoiyeneba:gamoiyeneba:

fluoresenciuri saRebavebifluoresenciuri saRebavebi

interkalirirebuli (ukuCarTviTi) interkalirirebuli (ukuCarTviTi) orjaWviani dnmorjaWviani dnm

modificirebuli dezoqsinukleotidebi, modificirebuli dezoqsinukleotidebi, romlebic fluoresencireben dnm romlebic fluoresencireben dnm komplementarul fragmentebTan komplementarul fragmentebTan hibridizaciis Semdeghibridizaciis Semdeg

pjr realur droSipjr realur droSi

multirezistentuli tuberkulozis multirezistentuli tuberkulozis swrafi diagnostikisaTvis myar swrafi diagnostikisaTvis myar niadagze niadagze miRebuli miRebuli M.Tuberculosis M.Tuberculosis kulturidan;kulturidan;

tuberkulozistuberkulozis swrafi swrafi diagnostikisaTvis diagnostikisaTvis uSualod masalidan (naxveli);uSualod masalidan (naxveli);

tuberkulozis mkurnalobis tuberkulozis mkurnalobis monitoringis efeqturobis monitoringis efeqturobis Sesafaseblad.Sesafaseblad.

pjr realurpjr realur droSidroSimultirezistentuli tuberkulozis swrafi multirezistentuli tuberkulozis swrafi

diagnostikadiagnostika

samizne jgufi: myar niadagze samizne jgufi: myar niadagze (LJ) (LJ) miRebulimiRebuli

196 196 M.Tuberculosis M.Tuberculosis kulturakultura

erTi testiT gamovlinda erTi testiT gamovlinda M.Tuberculosis M.Tuberculosis 25 25

genuri mutacia, romelbic genuri mutacia, romelbic pasuxismgebelni pasuxismgebelni

arian arian INH, RIF, STM, ETM INH, RIF, STM, ETM rezistentobazerezistentobaze

multirezistentuli tuberkulozis swrafi multirezistentuli tuberkulozis swrafi diagnostikadiagnostika

SedegebiSedegebi

     gamoyofili Stamebis gamoyofili Stamebis

raodenoba da Sedegbiraodenoba da Sedegbi

sensitiurosensitiurobaba

specifiurobspecifiurobaa

      INHINH RIFRIF SMSM EMBEMB

antituberkulozuantituberkulozuri preparatebiri preparatebi

fenotipurifenotipuri (lJ, DST) (lJ, DST) RR RR SS SS

genotipuri (genotipuri (Target-Target-Enriched Multiplex PCR)Enriched Multiplex PCR)

RR SS RR SS

INHINH    3535 66 66 149149 85.40%85.40% 96.30%96.30%

RIFRIF    1717 11 11 117117 94.50%94.50% 99.40%99.40%

SMSM    4848 2121 11 126126 89.60%89.60% 99.20%99.20%

EMBEMB    33 33 22 188188 50%50% 98.90%98.90%

M.Tuberculosis M.Tuberculosis swrafi diagnostika swrafi diagnostika uSualod masalidanuSualod masalidan

100 axali sadiagnozo SemTxveva100 axali sadiagnozo SemTxveva

kulturalurad dadebiTi 59 SemTxvevakulturalurad dadebiTi 59 SemTxveva

M.Tuberculosis M.Tuberculosis diagnostikisdiagnostikis meTodebis SedarebameTodebis Sedareba

98.30%

98.30%

86.40%

LJ PCR AFB

tuberkulozis mkurnalobis tuberkulozis mkurnalobis monitoringis efeqturobis Sefasebamonitoringis efeqturobis Sefaseba

samizne jgufi: 65 axali sadiagnozo samizne jgufi: 65 axali sadiagnozo SemTxvevaSemTxveva

masala Segrovda mkurnalobis dawyebidanmasala Segrovda mkurnalobis dawyebidan

2 kviris, 1, 2 da 4 Tvis Semdeg2 kviris, 1, 2 da 4 Tvis Semdeg

mgb baqterioskopuli gamokvleva (cil-mgb baqterioskopuli gamokvleva (cil-nilseni), daTesva myar niadagze nilseni), daTesva myar niadagze (LJ),(LJ), pjr pjr realur droSirealur droSi

83.10%

98%

75.00%

80.00%

85.00%

90.00%

95.00%

100.00%

AFB PCR

M.Tuberculosis M.Tuberculosis diagnostikisdiagnostikis meTodebis SedarebameTodebis Sedareba

120

8

77 81

0

20

40

60

80

100

120

C+ P+ C+ P- C-P+ C-P-

pjr realur droSi da daTesva pjr realur droSi da daTesva myar niadagze (myar niadagze (LJ) LJ)

((meTodebismeTodebis TanxmobaTanxmoba))

dadebiTobis maCveneblis dadebiTobis maCveneblis Semcireba mkurnalobis Semcireba mkurnalobis

monitoringis monitoringis drosdros

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

2 weeks 4 weeks 8 weeks 16 weeks

Culture

PCR

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

≤ 1 Month ≥2 Month

Non MDR

MDR

uaryofiTobis maCveneblis zrda uaryofiTobis maCveneblis zrda MDR MDR dada Non MDRNon MDR SemTxvevebSi mkurnalobis SemTxvevebSi mkurnalobis monitoringis dros (pjr da daTesva monitoringis dros (pjr da daTesva LJ)LJ)

tuberkulozis swrafi diagnostika uSualod tuberkulozis swrafi diagnostika uSualod masalidanmasalidan

multirezistentuli tuberkulozis swrafi multirezistentuli tuberkulozis swrafi diagnostikadiagnostika

maRali specifiuroba da sensitiuroba maRali specifiuroba da sensitiuroba RIF RIF da da INH INH rezistentobis ganmsazRvreli genis mimarTrezistentobis ganmsazRvreli genis mimarT

analizis Sedegs sWirdeba mxolod 2-4 saaTi analizis Sedegs sWirdeba mxolod 2-4 saaTi rogorc gamoyofili kulturidan aseve uSualod rogorc gamoyofili kulturidan aseve uSualod naxvelidannaxvelidan

daskvnadaskvna

Georgia - Vanderbilt University, (Nashville, Tennessee, USA), 2006-2008 Georgia - Vanderbilt University, (Nashville, Tennessee, USA), 2006-2008 CRDF project:CRDF project:"Monitoring Chemotherapy Regimen Efficacy by Mycobacterium "Monitoring Chemotherapy Regimen Efficacy by Mycobacterium Tuberculosis mRNA Detection in Sputum Specimens”.Tuberculosis mRNA Detection in Sputum Specimens”.

"Simultaneous Detection of Tuberculosis Drug Resistance Mutations by a Target-"Simultaneous Detection of Tuberculosis Drug Resistance Mutations by a Target-Enriched Multiplex PCR" - Enriched Multiplex PCR" - Abstract for the 107th General Meeting of the American Abstract for the 107th General Meeting of the American Society for microbiology, May 21-25, 2007, Toronto, CanadaSociety for microbiology, May 21-25, 2007, Toronto, Canada..

““Prevalence of and Molecular Basis for Tuberculosis Drug Resistance in the Republic of Prevalence of and Molecular Basis for Tuberculosis Drug Resistance in the Republic of Georgia: Validation of a Templex System for the Detection of Drug Resistance-Related Georgia: Validation of a Templex System for the Detection of Drug Resistance-Related Mutations" - Mutations" - Antimicrobial Agents and Chemotherapy. 52:725-9.USA. Antimicrobial Agents and Chemotherapy. 52:725-9.USA.

“ “Monitoring Therapy Efficacy by Real-Time Mycobacterium tuberculosis mRNA Monitoring Therapy Efficacy by Real-Time Mycobacterium tuberculosis mRNA Detection in Sputum Specimens” - Detection in Sputum Specimens” - Abstract for the 108th General Meeting of the Abstract for the 108th General Meeting of the American Society for Microbiology (June 1-5, 2008, Boston, MA), American Association American Society for Microbiology (June 1-5, 2008, Boston, MA), American Association for Clinical Chemistry (January 23, 2009, Washington, DC).for Clinical Chemistry (January 23, 2009, Washington, DC).

””DNA and mRNA-Targeted Real-Time TaqMan PCR Assays for Rapid DNA and mRNA-Targeted Real-Time TaqMan PCR Assays for Rapid Detection of Mycobacterium tuberculosis from Sputum Specimens” - Detection of Mycobacterium tuberculosis from Sputum Specimens” - Abstract Abstract for for 39th Union World Conference on Lung Health (16-20 October 200839th Union World Conference on Lung Health (16-20 October 2008 , Paris, , Paris, France).France).

gmadlobT yuradRebisTvis gmadlobT yuradRebisTvis