Nghiên cứu đặc điểm phân tử gen rpob, katg của vi khuẩn lao kháng đa thuốc ở việt nam

1 T VN Lao l mt nhng bnh truyn nhim hng u gõy t l t vong trờn th gii ngy Theo thng kờ ca T chc Y t Th gii (WHO), khong 1/3 dõn s th gii nhim lao Mi nm, cú thờm gn triu ngi mc lao mi v khong 1,3 triu ngi t vong lao Trong , 81 s bnh nhõn thuc v 22 nc c g nh n ng bnh lao [133] Trong , t l ph t hin m c dự liờn tc c ci thin nhng ch t 66 s bnh nhõn c tớnh [133], c nhng hn ch v cụng t c chn o n, c bit l lao kh ng thuc Nh vy, cũn rt nhiu bnh nhõn lao khụng c ph t hin v cha tr ang tip tc lõy lan cho cng ng Theo thng kờ ca WHO, Vit Nam l mt quc gia c t l bnh lao cao Theo b o c o ca Chng trỡnh Chng lao Quc gia (CTCLQG) nm 2013, tng s ngi mc lao l 190.000; ú, s ngi mc lao mi khong 130.000, s trng hp t vong lao c t nh l 17.000 ngi Tỡnh hỡnh kh ng thuc ca vi khun lao ti Vit Nam ang l mt ng lo ngi T l vi khun lao kh ng a thuc bnh nhõn iu tr li l 23 , bnh nhõn lao mi l 4% [4] Trong c c loi kh ng thuc ca vi khun lao, kh ng rifampicin v isoniazid - nhng thuc ch l c iu tr lao - c quan tõm nht vỡ thng dn n tht bi iu tr v t vong Theo nh ngha ca WHO, lao kh ng a thuc l trng hp kh ng vi t nht ng thi rifampicin v isoniazid [135] S kh ng vi thuc chng lao dũng ny c cho l c t bin trờn mt s gen liờn quan, c bit l c c gen rpoB v katG Cỏc nghiờn cu v t nh kh ng thuc ca vi khun lao cho thy, rt him c c chng kh ng rifampicin n thun m thng c t nht 90 c c chng lao lõm sng kh ng rifampicin ng thi cng kh ng isoniazid [15], [62] Nh vy, vic ph t hin c c chng lao kh ng rifampicin cng c ngha l x c nh c lao kh ng a thuc [92] Cỏc nghiờn cu v phõn t ca vi khun lao kh ng rifampicin ó cụng b cho thy, t nh kh ng rifampicin liờn quan n t bin gen rpoB, cũn kh ng isoniazid thng liờn quan n t bin gen katG [112], [140] Chn o n lao kh ng thuc ch yu d a vo phng ph p kh ng sinh v phi mt khong tun mi cho kt qu Chn o n nhanh vi khun lao kh ng thuc, c bit l kh ng a thuc bng k thut sinh hc phõn t l cn thit cụng t c gi m s t v kim so t lao C c kit thng mi ca nc ngoi chn o n lao kh ng thuc c gi thnh cao v thng ch nhm vo c c im t bin 526, 531 trờn gen rpoB v 315 trờn gen katG Cỏc nghiờn cu v lao kh ng thuc tng khu v c li ph t hin thờm c c im t bin v tr kh c nh 514, 533, 176 trờn gen rpoB; c bit tn s c c t bin thay i gia c c cng ng ngi v vựng a lý [15], [52], [65], [88], [110], [140] c im phõn t ca c c chng kh ng thuc phõn lp c t c c vựng kh c trờn th gii, nht l t c c quc gia c t l bnh lao v t l lao kh ng thuc cao s to iu kin thun li cho vic ph t trin c hiu qu hn c c phng ph p phõn t ph t hin nhanh ch ng t nh kh ng thuc phự hp vi tng nc v tng khu v c Vit Nam cha c c c d kin y v c im phõn t ca vi khun lao kh ng a thuc nc nờn cha c c s p dng nguyờn bn kit chn o n lao kh ng thuc ca nc ngoi Vỡ vy chỳng tụi th c hin ti: Nghiờn cu c im phõn t gen rpoB, katG ca vi khun lao khỏng a thuc Vit Nam nhm mc tiờu: nm 2008-2009 phõn l Vi N Chng T N QU N 1.1 Bnh lao Lao l cn bnh truyn nhim xut hin t lõu C c nghiờn cu du t ch ho thch cho thy, bnh lao xut hin ngi khong 400 - 230 nm trc Cụng nguyờn i dch lao bựng ph t chõu u th k XVII, lan sang chõu M v kộo di cho n tn th k XIX Trong giai on ny, nhng mụ t v gii phu, bnh lý, t nh cht lõy lan ca cn bnh ny ó c cụng b nh nhng cụng trỡnh nghiờn cu ca enjamin Martien (1720), Franciscus Sylvius de la Boe (1769) v Jean-Antoine Villemin (1865) Tỏc nhõn gõy bnh lao, Mycobacterium tuberculosis, ch nh thc c x c nh v mụ t vo nm 1882 õy l cụng trỡnh nghiờn cu ca Robert Koch (1843-1910) ghi nhn cụng lao ca ụng, ngi ta cũn gi tr c khun lao gõy bnh trờn ngi l tr c khun Koch ( acille de Koch) [92] Trong th k 20, bnh lao ó git cht khong 100 triu ngi T nm 1943, streptomycin, thuc chng lao u tiờn c phỏt hin v c s dng iu tr cú hiu qu Sau hng lot thuc chng lao kh c i ó m mt k nguyờn mi iu tr bnh lao, cng nh ó cu cha cho hng triu bnh nhõn lao cn bnh nguy him ny [92] Cựng vi s phỏt trin ca i dch HIV/AIDS, dch t lao ton cu ó bc sang mt hng phỏt trin mi gõy nhng thỏch thc vụ cựng to ln cho cuc chin chng lao ca loi ngi, l s tng t c gia lao v i dch HIV/AIDS v s phỏt trin ca cỏc chng lao khỏng thuc [92], [131], [133] 1.1.1 Tỡnh hỡnh bnh lao trờn th gii Hin nay, trờn th gii c khong 2,2 t ngi nhim lao (chim 1/3 dõn s th gii) Theo s liu cụng b ca T chc Y t Th gii (WHO) [133], c t nh nm 2012 c thờm khong 8,6 triu ngi mc lao mi (trung bỡnh 122 trng hp trờn 100 000 dõn) v 1,3 triu ngi t vong lao (trong , 940 000 ngi HIV (-) v 330 000 ngi t vong lao/HIV (+)) Trong s c c trng hp t vong, c 170 000 trng hp mc lao kh ng a thuc [133] Phõn b ton cu bnh lao nghiờng v ph a c c nc c thu nhp thp v nh m nn kinh t mi ni Hu ht c c trng hp xy chõu (58 ) v chõu Phi (27 ), mt t l nh hn ph a ụng khu v c a trung Hi (8 ), khu v c chõu u (4 ) Khong 81 s bnh nhõn lao ton cu thuc 22 nc c g nh n ng bnh lao cao [133] Trong tng s 8,6 triu ngi mc lao mi trờn ton cu nm 2012, c khong 13 sng chung vi HIV T l c c trng hp bnh lao ng nhim vi HIV cao nht c c quc gia thuc khu v c chõu Phi (82 ) [133] 1.1.2 Tỡnh hỡnh bnh lao Vit Nam Theo b o c o ca WHO nm 2012, Vit Nam ng th 12/22 nc c g nh n ng bnh lao cao trờn ton cu khu v c Tõy - Th i ỡnh Dng, Vit nam ng th ba sau Trung quc v Philippin v s lng bnh nhõn lao cng nh bnh nhõn lao mi xut hin hng nm [133] Nm 2013, t l mc lao mi c c th l 144/100.000 dõn; t l hin mc lao c c th l 209/100.000 dõn; t l t vong lao l 19/100.000 dõn [4] S liu ghi nhn ca chng trỡnh chng lao quc gia hng nm cho thy, tỡnh hỡnh bnh lao ngy cng din bin phc hn, t l ph t hin ca bnh trờn th c t c th thp hn s c t nh trc õy Mt s lng ng k c c bnh nhõn lao cha c chn o n v iu tr l ngun lõy quan trng cng ng iu s tng thờm s kh khn i vi cụng t c chng lao khụng nhng nhng nm ti m c th cũn thi gian kh di [2] Vi khun lao Vi khun lao c tờn khoa hc l Mycobacterium tuberculosis Th c cht t c nhõn gõy bnh lao l mt phc hp gm nhiu loi c gi chung l Mycobacterium tuberculosis complex (MT C) C c loi phc hp ny l t c nhõn gõy nờn bnh lao trờn c c vt ch kh c Kh nng gõy bnh ca c c loi l kh c nhau, loi gõy bnh thng xuyờn trờn ngi l Mycobacterium tuberculosis (MTB) m hỡnh th , cu trỳc Vi khun lao l tr c khun mnh, di 2-4 àm, rng 0,3-1,5 àm, ng riờng l ho c xp thnh hỡnh ch N, Y, V ho c thnh dóy phõn nh nh nh cnh cõy Tuy nhiờn, cng tựy thuc vo iu kin nuụi cy v thi gian nuụi cy m vi khun c k ch thc v hỡnh dng thay i, t cu tr c khun n tr c khun di [12], [92] Vi khun lao trờn kớnh hin vi quang hc Hỡ Hỡ Vi khun lao trờn kớnh hin vi in t * Ngun: theo Palomino J C.(2007)[92] Vi khun lao kh bt mu c c thuc nhum thụng thng Thng nhum theo phng ph p Ziehl - Neelsen, tr c khun lao khụng b cn v acid lm mt mu ca carbonfuchsin, bt mu [3], [12] Mycobacterium tuberculosis c cu trỳc v ch t bo phc tp, hon ho m t vi sinh vt c c Nú khụng nhng m bo cho s tn ti ca vi khun m cũn lm cho vi khun bn vng i vi hin tng th c bo, xõm nhp vo c th vi khun lao kh b tiờu dit Vỏch c vai trũ quan trng i vi kh nng gõy bnh ca vi khun Thnh t bo vi khun lao c t l lipit cao, chim ti hn 60 trng lng khụ ca thnh t bo, axit mycolic l thnh phn lipit ch yu Thnh t bo cng c c trng bi s liờn kt gia c c axit mycolic v c c cht lipid phc tp, to nờn c t nh ca vi khun lao, lm tng kh nng chng thm nc ca thnh vi khun giỳp vi khun tn ti lõu vi mụi trng bờn ngoi, chng kh nng b hu dit bi i th c bo v c c t bo dch Axit mycolic l c c cht bộo nh nh anpha nht tỡm thy thnh t bo ca Mycobacterium v Corynebacterium Chỳng chim 50 trng lng khụ ca v t bo Mycobacteria [92] m nuụi cy Vi khun lao a kh bt buc, khụng mc c iu kin k kh Vỡ vy, c c trng hp lao phi, thng tn thng hay g p thu trờn ca phi Tuy nhiờn, iu kin thiu oxy nh c c nt vụi ho , cc x ho , cc bó u rn chc vi khun lao tn ti trng th i khụng hot ng kh nhiu nm [3], [12], [92] Khụng nuụi c vi khun lao mụi trng thụng thng, phi nuụi mụi trng c bit, giu cht dinh dng, cha trng, khoai tõy, citrat, glycerol, asparagin, xanh malachit Thng dựng mụi trng c Lửwenstein c ci tin bi Jensen, ho c mụi trng lng Sauton [3], [12], [92] Vi khun lao l vi khun mc chm, thi gian mt th h ca Mycobacterium tuberculosis khong 18 gi, phi 4-6 tun sau mi hỡnh thnh khun lc in hỡnh, dng R [3], [92] Hỡ K ụ * Ngun: theo Lờ Vn Phng (2009)[9] ụ y m phõn t vi khun lao gen hon chnh ca Mycobacterium tuberculosis chng H37Rv c chiu di 4.411.532 bp, bao gm 3974 gen, c 3924 gen mó h a cho protein v 50 gen mó h a cho c c RN chc nng õy l genome cú kớch thc rt ln, cha nhiu Guanine v Cystosine (G + C khong 65 ) v khụng thay i hu nh trờn ton b genome [92] Mt nhng c t nh c quan tõm ca Mycobacterium tuberculosis l s c m t v phõn b ca c c trỡnh t chốn (insertion sequence IS), nh IS6110 v IS1081, c bit l on IS6110 Tuy nhiờn, mt s lng ng k c c chng khuyt gen IS6110 ó c tỡm thy chõu , n v Vit Nam [40], [70], [87] Gen 23S rDNA l on gen mó h a cho tiu phn 23S ca ribosome v c m t tt c c c chng lao gõy bnh Vỡ th, c c gen ny ang c quan tõm thit k kit chn o n vi khun lao Vi khun lao khỏng thuc Tỡ ỡ y Vi khun lao kh ng thuc ang l mt tr ngi ln i vi cụng t c phũng chng lao ton cu Nm 2008, t l lao kh ng a thuc l 3,6 T l kh ng vi c c thuc chng lao dũng trờn ton cu l 59 T l kh ng a thuc s lao mi thay i t th ph t thay i t n 28,3 Trng hp kh ng a thuc n 61,6 Ti Uzbekistan t l kh ng a thuc th ph t lờn n 60,0 , zerbaijan l 55,8 [131] Vi khun lao kh ng a thuc ang l mt th ch thc ln cho ton cu c t nh nm 2012 c 450.000 trng hp lao kh ng a thuc trờn ton th gii [133], chim 3,6 tng s mc lao mi v 20,2 s lao t i ph t Trung Quc, n , Liờn bang Nga l nhng nc c t l lao kh ng thuc v kh ng a thuc ln nht (chim trờn 50 s trng hp lao phi kh ng a thuc trờn ton cu) [133] i vi c c trng hp mc lao kh ng a thuc thỡ thng kh khn hn iu tr, liờn quan n t l mc v t l t vong cao, kộo di thi gian iu tr v lm tng nguy c lan truyn c c chng kh ng thuc cng ng Trong thuc chng lao hng u ch c thuc, thỡ thuc chng lao loi hai thng c c t nh cao v gi thnh rt t i vi lao kh ng a thuc m rng (XDR-T ) vic iu tr s kh khn v tn kộm hn nhiu so vi MDR-T v t l tht bi cao hn T l XDR-TB s MDR-T thay i t n 30 c c quc gia kh c trờn ton cu [131] Nhng ngi mc bnh lao vi chng vi khun kh ng a thuc m rng cn phi iu tr trờn nm so vi t - th ng trc õy v chi ph iu tr cng tng lờn gp 100 ln S MDR-TB ) 008 (b Khu vc S lng Chõu Phi 69 000 Chõu M 200 ụng a Trung Hi 24 000 Chõu u 81000 ụng Nam 130 000 Tõy Th i ỡnh Dng 120 000 Tng cng 440 000 * Ngun: theo WHO(2010) [131] Ti Vit Nam, tỡnh hỡnh kh ng thuc ca vi khun lao ang l mt ng lo ngi T l kh ng thuc c c trng hp lao mi l 32,7% v thuc loi cao trờn th gii [89] Nm 2013, t l lao kh ng a thuc bnh nhõn iu tr li l 23 , t l kh ng a thuc bnh nhõn mi l 4% Vit Nam ng th 14 s 27 nc c g nh n ng bnh lao kh ng a thuc trờn ton cu [4] T nh kh ng thuc ca vi khun lao c nh ngha l s gim mc mn cm ca Mycobacterium tuberculosis in vitro lm cho chỳng c s kh c bit vi c c chng di cha bao gi tip xỳc vi thuc khỏng sinh Trong c c trng hp lao kh ng thuc cn phi phõn bit [10], [131], [133], [135]: - Lao khỏng n thuc (monoresistant TB) l nhng trng hp vi khun lao ch kh ng vi mt thuc chng lao dũng th nht: isoniazid, rifampicin, pyrazinamide, ethambutol ho c streptomycin - Lao khỏng nhiu thuc (polydrug resistant TB): l nhng trng hp vi khun lao kh ng vi nhiu hn mt thuc chng lao dũng th nht (nhng khụng phi kh ng ng thi c isoniazid v rifampicin) - Lao khỏng a thuc (MDR-T ): l vi khun lao kh ng t nht vi c loi thuc l isoniazid v rifampicin - Lao khỏng a thuc m rng (XDR-T ): l s kh ng vi isoniazid v rifampicin cng vi fluoroquinolone v t nht s thuc chng lao dũng th hai dựng ng tiờm (amikacin, kanamycin ho c capreomycin) Kh ng thuc tiờn ph t l kh ng thuc xy mt bnh nhõn mi c chn o n lao, trc cha s dng mt liu ph p iu tr chng lao no ho c ó iu tr vi thi gian di th ng, cũn kh ng thuc th ph t l s ph t trin ca kh ng thuc ho c sau liu ph p iu tr bng ho cht t nht l 1th ng Kh ng thuc t nhiờn l t nh kh ng thuc ca c c chng kiu di vi c c thuc c hiu V d: tt c c c chng M bovis kiu di u c t nh kh ng thuc t nhiờn vi pyrazinamide, Mycobacterium tuberculosis cú t nh kh ng t nhiờn vi penicillin 33 C khỏng thuc ca vi khun lao T nh kh ng thuc ca vi khun lao liờn quan n s ph t sinh c c t bin mt c ch ngu nhiờn c c gen nm vựng nhõn ca t bo vi khun C c chng vi khun lao hoang di khụng tip xỳc vi c c thuc chng lao s khụng bao gi xut hin t nh kh ng thuc ngoi tr kh ng thuc t nhiờn ca 10 cỏc loi vi khun vi mt s thuc nh M bovis khỏng pyrazinamide, M tuberculosis khỏng penicillin, [94], [140] Hỡ C *Ngun: Zhang Y (2009) [140] Trong qu trỡnh nhõn lờn ca tr c khun lao, t nh kh ng thuc c ph t trin theo nhng trt t x c nh T nh kh ng thuc s ph t sinh ngu nhiờn c c t bin liờn quan ny c gi l t nh kh ng di truyn Khi khụng c m t ca thuc kh ng sinh, c c chng vi khun lao mn cm thuc ph t trin ln t c c chng kh ng thuc S c m t ca thuc kh ng sinh iu tr ó cung cp mt p l c chn lc cho c c chng vi khun lao C c chng mn cm b c ch sinh trng, thm ch b tiờu dit, c c chng kh ng thuc tr thnh u th hỡnh thnh t nh kh ng thuc thu c, c bit l c c bnh nhõn c cha mt lng ln tr c khun lao S lõy lan ca c c chng kh ng thuc ny sang nhng ngi kh c lm cho nhng ngi ny b nhim vi khun lao kh ng thuc t u (t nh kh ng thuc s cp) [94], [134], [140] T nh kh ng nhiu loi 122 55 Huong N T., Lan N T N, Cobelens F G J, et al (2006), "Antituberculosis Drug Resistance in the South of Vietnam: Prevalence and Trends", J Infect Dis, 194, pp 1226-1232 56 Huyen M N., Tiemersma E W., Lan N T., et al (2010), "Validation of the GenoTypeđ MTBDRplus assay for diagnosis of multidrug resistant tuberculosis in South Vietnam", BMC Infect Dis, doi:10.1186/1471-233410-149 57 Hwang H Y., Chang C Y., Chang L L., et al (2003), "Characterization of rifampicin-resistant Mycobacterium tuberculosis in Taiwan", J Med Microbiol, 52, pp 239-245 58 Iqbal S., Shabir I., Iqbal R., et al (2004), "Role of KatG Gene in Resistant Buildup Against Isoniazid (INH)- A first Line Anti-TB Drug", Int J Agri Biol, (3), pp 581-584 59 Isfahani B.N., Tavakoli A., Salehi M., et al (2006), "Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods", Mem Inst Oswaldo Cruz, 101 (6), pp 597-602 60 Jagielski T., rzeszczuk M., Kamiski M., et al (2013), "Identification and analysis of mutations in the katG gene in multidrug-resistant Mycobacterium tuberculosis clinical isolates", Pneumonol Alergol Pol , 81, pp 298-307 61 Jamieson F B., Guthrie J L., Neemuchwala A., et al (2014), "Profiling of rpoB Mutations and MICs for Rifampin and Rifabutin in Mycobacterium tuberculosis", J Clin Microbiol, 52 (6), pp 21572162 62 Johnson R., Streicher E M., Louw G E., et al (2006), "Drug Resistance in Mycobacterium tuberculosis", Curr Issues Mol Biol, 8, pp 97112 123 63 Kalokhe A S., Shafiq M., and Lee J C (2013), "Multidrug-resistant tuberculosis drug susceptibility and molecular diagnostic testing: a review of the literature", Am J Med Sci, 345 (2), pp 143-148 64 Kamachi S., Hirabayashi K., Tamoi M., et al (2015), "The crystal structure of isoniazid-bound KatG catalaseperoxidase from Synechococcus elongatus PCC7942", FEBS Journal, 282, pp 54-64 65 Kim B J., Lee K H., Park B N., et al (2001), "Detection of rifampinresistant Mycobacterium tuberculosis in sputa by nested PCR-linked single-strand conformation polymorphism and DNA sequencing", J Clin Microbiol, 39, pp 2610-2617 66 Kozhamkulov U., Akhmetova A., Rakhimova S., et al (2011), "Molecular Characterization of Rifampicin- and Isoniazid-Resistant Mycobacterium tuberculosis Strains Isolated in Kazakhstan", Jpn J Infect Dis, 64, pp 253-255 67 Khadka D K., Eampokalap B., Panitchakorn J., et al (2007), "Multiple mutations in katG and inhA identified in Thai isoniazid-resistant Mycobacterium tuberculosis isolates", Southeast Asian J Trop Med Public Health , 38 (2), pp 376-382 68 Khan S N., Niemann S., Gulfraz M., et al (2013), "Molecular Characterization of Multidrug-Resistant Isolates of Mycobacterium tuberculosis from Patients in Punjab, Pakistan", Pakistan J Zool., 45 (1), pp 93-100 69 Lavender C, Globan M, Sievers A, et al (2005), "Molecular Characterization of Isoniazid-Resistant Mycobacterium tuberculosis Isolates Collected in Australia", Antimicrob Agents Chemother, 49 (10), pp 4068-4074 124 70 Le T K., Bach K H., Ho M L., et al (2000), "Molecular fingerprinting of Mycobacterium tuberculosis strains isolated in Vietnam using IS6110 as probe", Tuber Lung Dis, 80 (2), pp 75-83 71 Lee A S., Lim I H., Tang L L., et al (2005), "High frequency of mutations in the rpoB gene in rifampin-resistant clinical isolates of Mycobacterium tuberculosis from Singapore", J Clin Microbiol, 43, pp 2026-2027 72 Lee A S., Lim I H K., Tang L L H., et al (1999), "Contribution of kasA analysis to detection of isoniazid resistant M tuberculosis in Singapore", Antimicrob Agents Chemother, 43, pp 2087 73 Ling D I., Zwerling A A., and M a P (2008), "Rapid diagnosis of drugresistant TB using line probe assays: from evidence to policy", Exp Rev Respir Med, 2, pp 583-588 74 Lingala M A L., Srikantam P., Suman J., et al (2010), "Clinical and geographical profiles of rpoB gene mutations in Mycobacterium tuberculosis isolates from Hyderabad and Koraput in India", J Microbiol Antimicrob, (2), pp 13-18 75 Luo T., Zhao M., Li X., et al (2010), "Selection of Mutations To Detect Multidrug-Resistant Mycobacterium tuberculosis Strains in Shanghai, China", Antimicrob Agents Chemother, 54 (3), pp 1075-1081 76 Mani C., Selvakumar N., Narayanan S., et al (2001), " Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis clinical isolates from India", J Clin Microbiol, 39, pp 2987-2990 77 Marahatta S.B., Gautam S., Dhital S., et al (2011), "katG (SER 315 THR) Gene Mutation in Isoniazid Resistant Mycobacterium tuberculosis", Kathmandu Univ Med J, 33 (1), pp 19-23 78 Martin A., Portaels F., and Palomino J C (2007), "Colorimetric redoxindicator methods for the rapid detection of multidrug resistance in 125 Mycobacterium tuberculosis: a systematic review and meta-analysis", J Antimicrob Chemother, 59, pp 175-183 79 Martớnez L M W., Castro G P., and Guerrero M I (2016), "A molecular platform for the diagnosis of multidrug-resistant and preextensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis", Mem Inst Oswaldo Cruz, Rio de Janeiro, 111 (2), pp 93100 80 Menon S., Dharmshale S., Chande C., et al (2012), "Drug resistance profile of Mycobacterium tuberculosis isolates to first line antituberculosis drugs: A five years study", Lung India, 29 (3), pp 227-231 81 Miller L P., Crawford J T., and Shinnick T M (1994), "The rpoB Gene of Mycobacterium tuberculosis", Antimicrob Agents Chemother, 38 (4), pp 805-811 82 Moaddab S R., Farajnia S., Kardan D., et al (2011), "Isoniazid MIC and KatG Gene Mutations among Mycobacterium tuberculosis Isolates in Northwest of Iran", Iran J Basic Med Sci, 14 (6), pp 540-545 83 Mokrousov I., Narvskaya O., Otten T., et al (2002), "High prevalence of KatG Ser315Thr substitution among isoniazid-resistant Mycobacterium tuberculosis clinical isolates from northwestern Russia, 1996 to 2001", Antimicrob Agents Chemother , 46, pp 1417-1424 84 Mokrousov I., Otten T., Vyshnevskiy B., et al (2003), "Allele-Specific rpoB PCR Assays for Detection of Rifampin-Resistant Mycobacterium tuberculosis in Sputum Smears", Antimicrob Agents Chemother, 47 (7), pp 2231-2235 85 Morgan M., Kalantri S., Flores L., et al (2005), "A commercial line probe assay for the rapid detection of rifampicin resistance in 126 Mycobacterium tuberculosis: a systematic review and meta-analysis", BMC Infect Dis, 5, pp 62 86 Musa H R., Ambroggi M., Souto A., et al (2005), "Drug susceptibility testing of Mycobacterium tuberculosis by a nitrate reductase assay applied directly on microscopy-positive sputum samples", J Clin Microbiol, 43, pp 3159-3161 87 Narayanan S., Das S, Garg R, et al (2002), "Molecular Epidemiology of Tuberculosis in a Rural Area of High Prevalence in South India: Implications for Disease Control and Prevention", J Clin Microbiol, 40 (12), pp 47854788 88 Negi S.S., Singh U., Gupta S., et al (2009), "Characterization of rpoB for detection of Rifampicin drug resistant by SSCP and Sequence analysis", Indian J Med Microbiol, 27 (3), pp 226-230 89 Nhung NV, Hoa NB, Sy DN, et al (2015), "The fourth national antituberculosis drug resistance survey in Viet Nam", Int J Tuberc Lung Dis., 19 (6), pp 670-5 doi: 10.5588/ijtld.14.0785 90 Orenstein E W., Basu S., Shah N S., et al (2009), "Treatment outcomes among patients with multidrug-resistant tuberculosis: systematic review and meta-analysis", Lancet Infect Dis, 9, pp 153-161 91 Osman M., Simpson J A., Caldwell J., et al (2014), "GeneXpert MTB/RIF Version G4 for Identification of Rifampin-Resistant Tuberculosis in a Programmatic Setting", J Clin Microbiol 52 (2), pp 635-637 92 Palomino J C., Leóo S.C., and Ritacco V (2007), Tuberculosis 2007: From basic science to patient WWW.TuberculosisTextbook.com care, Vol First Edition, 127 93 Palomino J C.,P AMaFa (2007), "Rapid drug resistance detection in Mycobacterium tuberculosis: a review of colourimetric methods", Clin Microbiol Infect, 13, pp 754-762 94 Parsons L M., Somoskoă A K., Gutierrez C., et al (2011), "Laboratory Diagnosis of Tuberculosis in Resource-Poor Countries: Challenges and Opportunities", Clin Microbiol Rev, 24 (2), pp 314350 95 Patra S K., Jain A (2012), "Molecular diagnosis of multidrug resistant tuberculosis", Int J Biomed Adv Research, (5), pp 273-280 96 Poudel A., Nakajima C., Fukushima Y., et al (2012), "Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis Isolated in Nepal", Antimicrob Agents Chemother, 56 (6), pp 28312836 97 Pozzi G., Meloni M., Iona E., et al (1999), "rpoB mutations in multidrug resistant strains of Mycobacterium tuberculosis isolated in Italy", J Clin Microbiol, 37, pp 11971199 98 Ramaswamy S., Musser J M (1998), "Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update", Tubercle and Lung Disease 79 (1), pp 3-29 99 Ramaswamy S.V., Reich R., Dou S.J., et al (2003), "Single nucleotide polymorphisms in genes associated with isoniazid resistance in Mycobacterium tuberculosis", Antimicrob Agents Chemother, 47, pp 1241-1250 100 Ramaswamy S V., Dou S J., Rendon A., et al (2004), "Genotypic analysis of multidrug-resistant Mycobacterium tuberculosis isolates from Monterrey, Mexico", J Med Microbiol, 53, pp 107-113 101 Reechaipichitkul W., Tubtim S., and Chaimanee P (2011), "Drug susceptibility patterns of Mycobacterium tuberculosis and clinical outcomes of drug-resistant tuberculosis at Srinagarind Hospital, a tertiary 128 care center in Northeastern Thailand", Southeast Asian J Trop Med Public Health, 42 (5), pp 1154-1162 102 Riahi F., Derakhshan M., Mosavat A., et al (2015), "Evaluation of point mutation detection in Mycobacterium tuberculosis with Isoniazid resistance using Realtime PCR and TaqMan probe assay", Appl Biochem Biotechnol, 175 (5), pp 2447-2455 103 Rindi L., Bianchi L., Tortoli E., et al (2005), "Mutations responsible for Mycobacterium tuberculosis isoniazid resistance in Italy", Int J Tuberc Lung Dis (1), pp 94-97 104 Sackett D.L., Haynes R.B., and Guyat G.H (1991), "Clinical Epidemiology: A basic science for clinical medicine", Boston: Little Brown, Second Edition (ISBN 0-136-76599-6) 105 Sajduda A., Brzostek A., awska M., et al (2004), "Molecular characterisation of rifampin-resistant Mycobacterium tuberculosis starins isolated in Poland", J Clin Microbiol 42, pp 2425-2431 106 Sarita Shah N., Moodley P., Babaria P., et al (2011), "Rapid Diagnosis of Tuberculosis and Multidrug Resistance by the MicroscopicObservation Drug-Susceptibility Assay", Am J Respir Crit Care Med, 183, pp 1427-1433 107 Schilke K., Weyer K., Bretzel G., et al (1999), "Universal pattern of rpoB gene mutations among multidrug-resistant isolates of Mycobacterium tuberculosis complex from Africa", Int J Tube Lung Dis, 3, pp 620-626 108 Shah N.S., Lan N.T., Huyen M.N., et al (2009), "Validation of the line-probe assay for rapid detection of rifampicin-resistant Mycobacterium tuberculosis in Vietnam", Int J Tuberc Lung Dis, 13 (2), pp 247-252 129 109 Sharma S., Madan M (2014), "Detection of Mutations in rpoB Gene of Clinically Isolated M tuberculosis by DNA Sequencing", J Mycobac Dis, (156), doi:10.4172/2161-1068.1000156 110 Sheng J., Li J., Sheng G., et al (2008), "Characterization of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis from eastern China", J Applied Microbiol, ISSN 1364-5072, pp.904-911 111 Shubladze N., Tadumadze N., and Bablishvili N (2013), "Molecular patterns of multidrug resistance of Mycobacterium tuberculosis in Georgia", Int J Mycobacteriology, 2, pp 73-78 112 Silva P E A D., Palomino J C (2011), "Molecular basis and mechanisms of drug resistance in Mycobacterium tuberculosis: classical and new drugs", J Antimicrob Chemother, doi:10.1093/jac/dkr173 113 Siqueira H.R., Freitas F.A.D., Oliveira D.N., et al (2009), "Isoniazidresistant Mycobacterium tuberculosis strains arising from mutations in two different regions of the katG gene", J Bras Pneumol, 35 (8), pp 773779 114 Siu G.K.H., Zhang Y., Lau T.C.K., et al (2011), "Mutations outside the rifampicin resistance-determining region associated with rifampicin resistance in Mycobacterium tuberculosis", J Antimicrob Chemother, 66, pp 730733 115 Somoskovi A., Parsons L M., and Salfinger M (2001), "The molecular basis of resistance to isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis", Respiratory Research, (3), pp 164-168 116 Titov L.P., Zakerbostanabad S., Slizen V., et al (2006), "Molecular characterization of rpoB gene mutations in rifampicine-resistant Mycobacterium tuberculosis isolates from tuberculosis patients in Belarus", Biotechnol J, 24, pp 1447-1452 130 117 Torres M.J., A C, Gonzalez N., et al (2002), "Rifampin and isoniazid resistance associated mutations in Mycobacterium tuberculosis clinical isolates in Seville, Spain", Int J Tuberc Lung Dis, 6, pp 160-163 118 Tho D Q., Ha D T M., Duy P M., et al (2008), "Comparison of MAS-PCR and GenoType MTBDR assay for the detection of rifampicinresistant Mycobacterium tuberculosis", Int.J Tuberc Lung Dis., 12 (11), pp 13061312 119 TracevskaT., Jansone I., Broka L., et al (2002), "Mutations in the rpoB and katG Genes Leading to Drug Resistance in Mycobacterium tuberculosis in Latvia", J Clin Microbiol, 40 (10), pp 3789-3792 120 Traore H., van Deun A., Shamputa I C., et al (2006), "Direct Detection of Mycobacterium tuberculosis-complex DNA and Rifampin Resistance in Clinical Specimens from Tuberculosis Patients by the Line Probe Assay; a Large Scale Study", J Clin Microbiol, 44, pp 4384-4388 121 Unissa A N., Selvakumar N., Narayanan S., et al (2015), "Investigation of Ser315 Substitutions within katG Gene in IsoniazidResistant Clinical Isolates of Mycobacterium tuberculosis from South India", BioMed Research International, 2015 (ID 257983), http://dx.doi.org/10.1155/2015/257983 122 Valvatne H., Syre H., Kross M., et al (2009), "Isoniazid and rifampicin resistance-associated mutations in Mycobacterium tuberculosis isolates from Yangon, Myanmar: implications for rapid molecular testing", J Antimicrob Chemother, 64, pp 694-701 123 Van Deun A., Martin A., and Palomino J C (2010), "Diagnosis of drug-resistant tuberculosis: reliability and rapidity of detection", Int J Tuberc Lung Dis, 14 (2), pp 131140 124 van Doorn H.R., de Haas P.E., Kremer K., et al (2006), "Public health impact of isoniazid-resistant Mycobacterium tuberculosis strains 131 with a mutation at amino-acid position 315 of katG: a decade of experience in The Netherlands", Clin Microbiol Infect, 12, pp 769-775 125 Van Soolingen D., de Hass P.E., van Doom H.R., et al (2000), "Mutations at amino acid position 315 of the katG Gene are Associated with High-level Resistance to isoniazid, Other Drug resistance, and successful transmission Mycobacterium tuberculosis in the Netherlands", J Infect Dis, 182, pp 1788-1790 126 Varela G., Gonzalez S., Gadea P., et al (2008), "Prevalance and dissemination of the Ser315Thr substitution within the katG enzyme in isoniazid- resistant strains of Mycobacterium tuberculosis in Uruguay", J Med Microbiol, 57, pp 1518-1522 127 Veluchamy M., Madhavan R., Narayanan S., et al (2013), "katG Gene as a Surrogate Molecular Marker Leading to Cause Drug Resistance in Mycobacterium tuberculosis Isolates", Am J Infect Dis and Microbiol, (5), pp 86-91 128 Wengenack N L., Uhl J R., St Amand A L., et al (1997), "Recombinant Mycobacterium tuberculosis KatG (S315T) is a competent catalase-peroxidase with reduced activity toward isoniazid", J Infect Dis, 176, pp 722-727 129 World Health Organization (2008), " Policy Statement Molecular Line Probe Assay for Rapid Screening of Patients at Risk of Multidrugresistant (MDR) TB)", Geneva WHO 2008 Available at: http://www.who.int/tb/dots/laboratory/policy/en/print.html" 130 World Health Organization (2010), Framework for Implementing New Tuberculosis Diagnostics 131 World Health Organization (2010), Multidrug and extensively drugresistant TB (M/XDR-TB): 2010 global report on surveillance and response 2010, WHO/HTM/TB/2010.3 132 132 World Health Organization (2011), Automated Real-time Nucleic Acid Amplification Technology for Rapid and Simultaneous Detection of Tuberculosis and Rifampicin Resistance: Xpert MTB/RIF System Policy Statement, WHO/HTM/TB/2011.4 133 World Health Organization (2013), Global Tuberculosis Report 2013, WHO report 2013 134 World Health Organization (2014), Companion handbook to the WHO guidelines for the programmatic management of drug-resistant tuberculosis, WHO/HTM/TB/2014.11 135 World Health Organization (2014), "Definitions and reporting framework for tuberculosis 2013 revision (updated December 2014)", WHO/HTM/TB/2013.2 136 Yu H (2007), Structural Studies of Mycobacterium tuberculosis katG, an INH drug activator, and brucella abortus VIRB11,and atpase of type IV translocation system, Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of doctor phylosophy 137 Zaczek A., Brzostek A., Augustynowicz-Kopec E., et al (2009), "Genetic evaluation of relationship between mutations in rpoB and resistance of Mycobacterium tuberculosis to rifampin", BMC Microbiol, 9:10 doi:10.1186/1471-2180-9-10 138 Zhang M., Yue J., Yang Y., et al (2005), "Detection of Mutations Associated with Isoniazid Resistance in Mycobacterium tuberculosis Isolates from China", J Clin Microbiol, 43 (11), pp 5477-5482 139 Zhang Y., Heym B., Allen B., et al (1992), "The catalaseperoxidase gene and isoniazid resistance of Mycobacterium tuberculosis", Nature, 358, pp 591-593 133 140 Zhang Y., Yew W W (2009), "Mechanisms of drug resistance in Mycobacterium tuberculosis", Int J Tuberc Lung Dis, 13 (11), pp 1320 1330 141 Zhao X., Yu H., Yu S., et al (2006), "Hydrogen peroxide-mediated isoniazid activation catalyzed by Mycobacterium tuberculosis catalaseperoxidase (KatG) and its S315T mutant", Biochemistry, 45, pp 41314140 142 Zhou A., Nawaz M., Duan Y., et al (2011), "Molecular Characterization of Isoniazid-Resistant Mycobacterium tuberculosis Isolates from Xian, China , Microb Drug Resist, 17 (2), pp 275-281 DOI: 10.1089/mdr.2010.0135 134 MC LC NHNG CH VIT TT D NH MC CC HèNH D NH MC CC NG D NH MC IU i iii iv v T VN Chng T N QU N 1.1 Bnh lao 1.1.1 Tỡnh hỡnh bnh lao trờn th gii 1.1.2 Tỡnh hỡnh bnh lao Vit Nam Vi khun lao 1.2.1 c im hỡnh th, cu trỳc 1.2.2 c im nuụi cy 1.2.3 c im phõn t vi khun lao Vi khun lao khỏng thuc 1.3.1 Tỡnh hỡnh kh ng thuc ca vi khun lao hin 1.3.2 Phõn loi kh ng thuc ca vi khun lao 1.3.3 C ch kh ng thuc ca vi khun lao 1.3.3.1 Khỏng rifampicin 11 1.3.3.2 Khỏng isoniazid 12 Cỏc phng phỏp chn oỏn lao khỏng thuc 13 1.4.1 Phng ph p x c nh kiu hỡnh 14 1.4.2 Phng ph p x c nh kiu gen 15 1.5 Nghiờn cu giỏ tr chn oỏn ca cỏc phng phỏp sinh hc phõn t chn oỏn vi khun lao khỏng a thuc 18 1.5.1 Trờn th gii 18 1.5.2 Ti Vit Nam 21 1.6 Gen rpoB v katG liờn quan n khỏng thuc vi khun lao 24 1.6.1 Gen rpoB 24 1.6.2 Gen katG 26 1.7 Nghiờn cu c im phõn t gen rpoB v katG cỏc chng vi khun lao 28 1.7.1 Trờn th gii 28 1.7.2 Tỡnh hỡnh nghiờn cu ti Vit Nam 31 Chng I TN V PHN PHP N HIấN CU 34 135 i tng nghiờn cu 34 a im nghiờn cu .34 2.3 Vt liu v thi t b nghiờn cu 35 2.3.1 C c sinh phm ho cht ch nh 35 2.3.2 C c m y v thit b ch nh 36 2.3.3 C c c p mi dựng nghiờn cu 37 Phng phỏp nghiờn cu 37 2.4.1 Thit k nghiờn cu 37 2.4.2 Phng ph p thu thp s liu 38 2.5.1 K thut kh ng sinh ph t hin vi khun lao kh ng thuc 40 2.5.2 T ch chit DN t c c chng vi khun nghiờn cu 41 2.5.3 K thut PCR nhõn on DN 42 2.5.4 Tỏch dũng gen rpoB v katG phc v cho gii trỡnh t 45 2.5.5 c trỡnh t DN trờn m y c trỡnh t t ng phõn t ch kt qu bng c c phn mm chuyờn dng 49 Xỏc nh giỏ tr ca phng phỏp sinh hc phõn t chn oỏn vi khun lao khỏng thuc 50 2.7 Phng phỏp phõn tớch v x lý s liu 51 Chng K T QU N HIấN CU 52 c im phõn t gen rpoB v gen katG ca cỏc chng vi khun lao khỏng thuc 52 3.1.1 Kt qu thu thp v v l a chn chng nghiờn cu .52 3.1.2 c im phõn t gen rpoB 54 3.1.3 c im phõn t gen katG 64 iỏ tr ca phng phỏp sinh hc phõn t chn oỏn vi khun lao khỏng a thuc 72 3.2.1 c t nh kh ng thuc ca c c chng vi khun lao thu thp c 72 3.2.2 Gi tr ca phng ph p sinh hc phõn t chn o n vi khun lao kh ng thuc 74 Chng BN LU N 77 c im phõn t vi khun lao khỏng thuc trờn gen rpoB v katG 77 4.1.1 Kt qu thu thp v l a chn chng nghiờn cu 77 4.1.2 c im phõn t vi khun lao kh ng thuc trờn gen rpoB 77 4.1.2.1 S lng v t l t bin trờn gen rpoB c c chng vi khun lao khỏng rifampicin 77 4.1.2.2 C c v tr t bin trờn gen rpoB c c chng vi khun lao kh ng rifampicin 80 136 4.1.2.3 T l v kiu t bin vựng RRDR trờn gen rpoB 84 4.1.2.4 C c v tr v kiu t bin mi trờn gen rpoB c c chng vi khun lao khỏng rifampicin 87 4.1.2.5 C c v tr v kiu t bin ngoi vựng RRDR 88 4.1.2.6 C c kiu kt hp v tr t bin 89 91 4.1.3.1 S lng v t l t bin trờn gen katG c c chng vi khun lao khỏng Isoniazid 91 4.1.3.2 t bin codon 315 trờn gen katG (315katG) 93 4.1.3.3 C c t bin kh c ngoi codon 315 trờn gen katG 97 4.1.3.4 T l t bin c c chng kh ng n vi isoniazid v kh ng a thuc 98 4.1.3.5 S lng v tr t bin trờn on gen katG c gii trỡnh t 99 4.1.3.6 C c kiu t bin trờn gen katG 100 iỏ tr ca phng phỏp sinh hc phõn t chn oỏn vi khun lao khỏng a thuc 101 4.2.1 c t nh kh ng thuc ca c c chng vi khun lao thu thp c 101 4.2.2 Gi tr ca phng ph p sinh hc phõn t chn o n vi khun lao kh ng a thuc 105 4.2.3 xut phng ph p sinh hc phõn t chn o n lao kh ng thuc 107 K T LU N 112 c im phõn t gen rpoB v katG vi khun lao khỏng thuc 112 1.1 c im phõn t gen rpoB vi khun lao kh ng thuc .112 1.2 c im phõn t gen katG vi khun lao kh ng thuc 112 iỏ tr ca phng phỏp sinh hc phõn t chn oỏn vi khun lao khỏng a thuc 113 KI N N H 114 D NH MC CC BI BO CễN LU N N TI LIU TH M KH O PH LC B LIấN QU N N TI ... chẩn đo n lao kh ng thuốc nước Vì th c đề tài: Nghiên cứu đặc điểm phân tử gen rpoB, katG vi khuẩn lao kháng đa thuốc Vi t Nam nhằm mục tiêu: ố ủ ủ ẩ ủ ẩ ượ năm 2008-2009 phân lậ ố Vi N ẩ ... n vi khuẩn lao Vi khuẩn lao kháng thuốc Tì ì ố ủ ẩ ệ y Vi khuẩn lao kh ng thuốc trở ngại lớn công t c phòng chống lao toàn cầu Năm 2008, tỷ lệ lao kh ng đa thuốc 3,6 Tỷ lệ kh ng với c c thuốc. .. khuẩn lao kháng đa thuốc Vi c nghiên cứu ph t c c đột biến gen rpoB katG liên quan kháng rifampicin, isoniazid kh ng đa thuốc c c chủng vi khuẩn lao giới Vi t Nam thường gắn liền với vi c đ nh