LTNG DUNG KI THUAT PHAN LTNG CHUOI POLYMERASE VA GIAI TRINH TLT GENE TRONG CHAN DOAN DINH DANH NGUYEN NHAN VIEM MU
NOI NHAN NOI SINH DO V I KHUAN
r DdT&n',B5Nhir Hem', Pham Hong Nhung" j
TOM TAT
Muc tieu: Nham buoc ddn ddnh gid hieu qud cua Id thudt polymerase (PCR) vd gidi trinh tu gene trong chdn dodn dinh danh vi khudn (VK) gdy benh a nhung mdu henh phdm Idy a nhung benh nhdn (BN) nghi nga viem mu noi nhdn noi sinh do VK (VMNNNSVK).
Phu'ffng phap: Nghien ciru mo td khong co doi chung. Diing kT thudt PCR vd gidi trinh tu de phdn tich 23 benh phdm dich kinh a 23 BN co trieu chung Idm sdng dien hinh cua VMNNNSVK co so sdnh voi ket qud cua nhuom soi vd nuoi cdy. Cdc thir nghiem duoc tiin hdnh tgi khoa Vi sinh, trudng Dgi hoc Gifu, Nhdt Bdn, thdng 1/2009.
Ket qua: Ket qud nhuom soi: 8/23 (34,8%) truong hop (TH) cho ket qud dm tinh, trong khi do PCR duong tinh d tdt cd cdc TH. Tren 10/23 (43,5%) nhuom soi quan sdt thdy nhieu logi VK, nhimg chi co 3 TH trong so ndy duoc khdng dinh tren PCR, 7 TH con lgi gidi trinh tu chi cho ket qud cua 1 logi VK. Ket qud nuoi cay: nuoi cdy cho ti le duong tinh thdp (3/23 - 13% ) . 3 TH duong tinh ndy deu triing khop voi kit qud ciia PCR vd gidi trinh tu sau ndy. PCR vd gidi trinh tu cho thdy gdn V2 (11/23) cdc TH Id phe cdu (Streptococcus pneumoniae). Co 5 TH phdt hien nhieu logi VK nhung do kho khdn ve Id thudt, chiing toi khong Idm duoc
tdch ddng de xdc dinh timg logi VK. Cdc TH cdn lgi gom: Enterococcus casseliflanis (1), Streptococcus sp (1), Staphylocuccus sp (1), S.heamolyticus (1), Bacillus sp (1), S. sciuri (1), Enterobacter hormaecher (1).
Kit luan: PCR vd gidi trinh tu cho ket qua dinh danh VK chinh xdc hon nhuom soi vd nuoi cdy, giiip ich cho dieu tri, dong thoi gop phdn xdc dinh 6 nhiem triing nguyen phdt cua benh VMNNNSVK.
Tir khoa: Viem mii noi nhdn noi sinh, PCR, giai trinh tugene.
I. DAT V A N D E
VMNNNSVK la mot benh li viem nhiem nang ne a cac mo va dich noi nhan do sir xam nhap ciia vi khuan tu co quan khac qua duong mau den mat. Day la mpt benh li cap cuu co the gay ton hai Ion ve chuc nang thi giac, tham chi co the phai bo nhan cau mac dii da dugc dieu tri tich cue.
'Benh vien Mai Trung uxnig,
"Khoa Vl sinh, Dal hoc Y Ha Noi
VK khi den mat (co quan dich) can co dieu kien thuan lgi de phat trien. Dieu kien thuan lgi do la sir suy giam siic de khang ciia ca the. VMNNNS theo y van thudng gap a nhung BN suy giam mien dich hoac suy kiet do cac benh li toan than. Tuy nhien, tren thuc te lam sang 6 Viet Nam, chiing toi thuong gap VMNNNS tren nhiing BN tre, khoe manh. Cho den nay, van chua co loi giai thich thoa dang cho hien tugng lam sang nay. Mgt trong nhung kho khan gap phai trong qua trinh dieu tn
NGHIEN CUtJ KHOA HOC
la chan doan ngm en nhan cua benh. Viec xac dinh tac nhan gay benh co tam quan trgng to Ion, quyet dinh hieu qua cua qua trinh dieu tri va tien lugng chiic nang cua benh. Cho den nay, cac xet nghiem vi sinh kinh dien thuong lam la nhuom soi va nuoi cay VK, nhung ti le duong tinh thap thay doi tir 21 - 63%. Dieu nay co the dugc giai thich nhu sau: (1) BN thuang nhap \ien kha muon sau khi da dugc dieu tn rat nhieu bang khang sinh truoe do (toan than va tai cho); (2) moi truang nuoi cay chua dat chuan, hien chua co moi truang nuoi ca>
yem khi. Thuc te lam sang na}' cho thay. nhu cau thiet yeu cua viec su dung mgt loai xet nghiem khac CO do nhay va do dac hieu cao hon. tra loi ket qua nhanh hon.
Tir 10 nam tra lai da}, nhieu tac gia tren the giai da thong bao ve su dung ki thuat PCR (polymerase chain reaction) va giai trinh tir (sequencing) trong phan tich dich ngi nhan lay tir BN nghi nga VMNN. KT thuat na} co the khac phuc dugc cac nhugc diem cua kT thuat phan lap va nuoi cay kinh dien: (1) chi can lugng benh pham it; khong doi hoi benh pham phai chira sinh vat con song (nghTa la phan tich dugc ca nhiing benh pham cua nhirng BN da dugc dieu tri khang sinh tnrdc do); tra ket qua kha nhanh (trong vong khoang 24 gio sau khi la} benh pham).
n. D 6 I Tl/ONG VA FHirONG PHAP NGHIEN ClTU 1. Doi tuong
23 benh pham dich kinh a 23 BN (nam dieu trj tai khoa Glocom. Benh \ ien Mat Trung uong tir thang 4/2008 den 11/2008) co trieu chimg lam sang di^n hinh cua VMNNNSVK.
2. Phuong phap
Nghien cim mo ta khong co doi chimg. Dimg kT thuat PCR \ a giai trinh tu de phan tich 23 benh pham dich kinh a 23 BN, co so sanh vdi ket qua ciia nhuom soi \a nuoi cay. Cac thu nghiem dugc tien
hanh tai khoa Vi sinh. trudng Dai hgc Gifii, Nhat Ban \ a o thang 1/2009.
Quy trinh lay benh phdm:
- Tat ca BN deu dugc lay benh pham trong phdng md theo qu}' trinh gidng nhau. Sat khuan thudng qu}" bang po\idone 5% va gay te canh nhan cau bang lidocaine 2%.
- Benh pham dich kinh la}- bang ma} cat dich kinh: trude khi mo dudng tr} en vao. hut benh pham qua dau cat dich kinh chia \ ao 2 xi-lanh. mdi xi-lanh la}' 0.2 - 0,3ml, mgt dimg cho cac xet nghiem vi sinh kinh dien (nhudm soi va nudi cay), mgt diing cho PCR va giai trinh tir.
- Benh pham dimg cho xet nghiem vi sinh kinh dien: sau khi lay se dugc cho soi tuoi. true tiep co nhudm gram de xac dinh so bd loai tac nhan ga}
benh (VK. nam ha} cac tac nhan khac) va nuoi ca}
vao cac mdi trudng thich hgp. Cac tac nhan khdng phai VK se dugc loai ra khoi nghien cim. VK dugc ca}' chu}'en vao mdi trudng thach sdcdla 37°C dugc lam giau them bang dich thioglycolate. Nudi cay ki khi a mdi trudng canh thang thioghcate hoac thach mau ki khi dugc lam giau vdi hemin va vitamin K a37°C.
Quy trinh phdn tich PCR vd gidi trinh tu:
- Benh pham dimg cho sinh hgc phan tu se dugc luu giir trong dieu kien vd khuan o nhiet do -20°C tnrdc khi su dung.
- Tach chiet DNA: Quy trinh tach chiet DNA va PCR dugc thue hien tai khoa Vi sinh, Dai hoc Gifii, Nhat Ban. DNA dugc tach chiet bang kit MORA (AMR, Gifii, Japan) theo hudng dan cua nha san xuat.
- Phan ling chuoi pohmerase (PCR): lam theo qu} trinh kT thuat chuan cua Kar}- Mullis.
- Vimg DNA dugc chgn nhan len de xac djnh VK la doan nam trong rRNA. Day la -vimg dugc bao tdn a cac loai VK va cd nhieu phien ban trong 1 t^ bao VK. Trong nghien cim na}. dau tien. de
nhan doan DNA chung tdi diing cap mdi chung cho tat ca cac loai VK (universal primer) gan vao don vi 16S cua rRNA. Chiing tdi sir dung cap mdi 8UA va 1485B, chu trinh nhiet cd dugc thay doi (94°C/5 phut; 40 chu ki gom 94°C/30giay, 55°C/60 gia}', va 72°C/60 giay, ket thuc bang 72°C/6 phiit) so vdi md ta ciia Brosius va cdng sir de khuech dai doan dai khoang 1500bp ma hda cho tieu phan 16S rRNA [1]. Tat ca cac lan lam PCR vdi benh pham ludn ludn cd chirng am va chimg duong chay cimg de loai, trir hien tugng am tinh va duong tinh gia.
- Trong nghien ciru, 5|al benh pham dugc dua true tiep vao phan img PCR. Neu phan irng PCR am tinh thi se can nhac den kha nang cd chat ire che ciia phan irng PCR trong benh pham. Tuy nhien, cho den nay ngudi ta van khdng xac dinh dugc chat lie che PCR trong benh pham dich kinh [6], [7].
Trong TH nay chung tdi tien hanh pha loang benh pham bang nude eat vd khuan theo ti le 50% (1:2), 20% (1:5), 10% (1:10) va 5% (1:20) sau do se lam lai phan irng dong thdi. Vdi bien phap nay chung tdi da loai bd dugc su ire che d tat ca cac mau benh pham (23 mau).
HI. KET QUA
Ket qua nghien ciru dugc tdng hgp d bang 1:
Bdng 1. Tong
- Giai trinh tu DNA: Cac san pham PCR sau khi dugc khuech dai, dugc giai trinh tu bang he thong may giai trinh tu ddng PRISM 3100 Genetic Analyzer (Applied Biosystems) vdi sinh pham BigDye™ Terminator v3.1 Cycle Sequencing Ready Reaction Kit theo hudng dan cua nha san xuat. Trinh tu cua doan gen ma hda cho 16S rRNA dai khoang lOOObp, dugc phan tich tren website ciia NCBI de dinh danh chimg VK cd mat trong benh pham mu ndi nhan.
- Nhirng chiing VK dugc xac dinh la Streptococus pneumoniae dua tren phan tich trinh tu ciia gen 16S rRNA se dugc tien hanh lam PCR khuech dai doan gen dnaJ ma hda cho Hsp40. Va giai trinh tu de khang dinh chac chan ten VK d miic dp loai, vi phan tich trinh tu ciia gen 16S rRNA chua du de xac dinh chinh xac miic do loai cho S. pneumoniae.
- Tach ddng san pham cua phan ung PCR (cloning), de xac dinh su cd mat cua cae loai khac nhau trong cimg mdt benh pham khdng dugc tien hanh do khdng du thdi gian va kinh phi. Nhiing benh pham nay dugc ghi ket qua la "mix" (hdn hgp).
hop dif lieu
Stt 1 2
4 5 6 7 8 9 10
Ho va ten N.D.N
L.PC L.TH N.VV L.T.N.Q
V.VH T V L B P T V T B T
N T . L Tuoi
8 4 3 32
1 40 51 4 4 29
Gioi Nam Nir Nu Nam
Nir Nam Nam Nu Nu Nir
Ngay lay 17/10/08
10/9/08 13/9/08 10/7/08 10/7/08 10/6/08 10/4/08 10/4/08 13/10/08 23/11/08
Nhuom soi CK+, TK+
CK+
CK+, TK+
TK+, TK- CK+, TK- CK+, TK+
(-) CK+. TK- CK+. TK-
(-)
Nuoi cay (-) S. pneumoniae
(-) (-) (-) (-) (-) (-) Bacillus spp
(-)
PCR & Sequencing S. pneumoniae
S. peumoniae S. pneumoniae S. pneumoniae S. pneumoniae
Mix
Staphylococcus sciuri S. pneumoniae
Bacillus spp Mix
NGHIEN CUtJ KHOA HOC
Stt 11 12 13 14 15 16 17 18 19 20 21 22 23
Ho va ten N. T T H
N B A K.TT N V. G
N H T D . T C N M T
T T H TK.H N.TT T T T N.VC D D T
Tuoi 1.5
4 25
5 30 72 65 43 30 68 4 4 22
Gioi Nu Nam Nam Nam Nam Nir Nam Nam Nam Nir Nir Nam Nam
Ngay lay 16/9/08 22/9/08 28/10/08
17/5/08 21/9/08 27/10/08 25/10/08 30/7/08
11/4/08 14/10/08 28/10/08 21/10/08 11/9/08
Nhuom soi TK+
CK+
CK+. TK- (-) (-) (-) CK+
CK+, TK+
(-) CK+, TK+
TK- (-) (-)
Nuoi cay (-) Streptococcus sp
(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-)
PCR & Sequencing S. pneumoniae Streptococcus sp Staphylococcus sp
S. pneumoniae Mix S. pneumoniae Staph} locoecus haemol}ticus
Mix S. Pneumoniae
Mix Enterobacter
hormaecher S. pneumoniae
Enterococcus casseliflanis Ket qud xet nghiem kinh dien:
- Ket qua nhudm soi: 8/23 (34,8%) TH cho ket qua am tinh, trong khi dd PCR duong tinh d tat ca cac TH. Tren 10/23 (43.5%) nhudm soi quan sat thay nhieu loai VK, nhung chi cd 3 TH trong so nay dugc khang dinh tren PCR. 7 TH cdn lai giai trinh tu chi cho ket qua cua 1 loai VK.
- Ket qua nudi ca}: nudi cay cho ti le duong tinh rat thap (3/23 - 13% cac TH). 1 TH cd Streptococcus pneumoniae. 1 TH la Bacillus spp., va TH cdn lai cho Streptococcus spp. 3 TH duong tinh nay deu tning khdp vdi ket qua cua PCR va giai trinh tu sau nay.
Ket qud cua PCR vd gidi trinh tir:
Gan Vl (11/23) cac TH cho ket qua la phe cau (Streptococcus pneumoniae). Cd 5 TH phat hien nhieu loai VK. nhung do khd khan ve kl thuat chiing tdi khdng lam dugc tach ddng de xac dinh timg loai VK. Ket qua eua PCR va giai trinh tu dugc tdm tiit vao bang 2. (Tat ca cac TH S. pneumoniae deu dugc dinh danh bang phan tich trinh tu eiia ca gen 16S rRNA va dnaJ).
Bdng 2. Ket qud PCR vd gidi trinh tir
Loai vi khuan S. pneumoniae Mix
Enterococcus casseliflanis
So lu-ffng (%) 11(47,8%)
5 (21,7%) 1 (4,3%)
Dac dilm va ca quan hay gay benh CK+, ki khi dung nap, ha} ga} benh d dudng hd hap
CK+, ki khi tuy tien, hgi sinh d dudng rudt, gay nhiem tning tiet nieu, mau, mang nao
Loai vi khuan
Streptococcus sp Staphylocuccus sp S. heamolyticus Bacillus sp S. sciuri
Enterobacter hormaecher
S6 lu-ong (%)
1 (4,3%) 1 (4,3%) 1 (4,3%) 1 (4,3%) 1 (4,3%) 1 (4.3%)
Dac diem va co quan hay gay benh
CK+, nhieu chung khac nhau, ga} benh a he hd hap. mau.
CO, mang nao
Tu cau Gr(+), hay ga} benh da. niem mac
Tu cau khdng tan hm'et. ha} ga}' viem md mem a BN suy giam mien dich
TK+, cd trong khdng khi va mdi trudng xung quanh Hg tu cau. ha}' gay nhiem tning tiet nieu
TK-, ki khi tuy tien, gay benh a dudng tieu hoa va tiet nieu
IV. BAN LUAN
Trong VMNN ndi chung va VMNNNS ndi rieng. nudi cay VK (NCVK) van dugc coi la tieu chuan vang trong chan doan va la mdt cdng cu hd trg rat dac lire cho dieu tri. Bdi khi NCVK duong tinh. khdng nhirng cho phep xac dinh loai VK ga}
benh ma cdn cho phep lam khang sinh dd va giiip cho viee hieu chinh dieu tri theo khang sinh do. Tuy nhien, ket qua NCVK d trong dieu kien hien tai a Viet Nam dat ket qua fliap (13% - 22,2%) [4]. Ti le duong tinh thap cd the do nhieu nguyen nhan:
do benh pham it. loang, do da sii dung khang sinh trude do, do VK ket d cac be mat cimg nhu the thu} tinh nhan tao hoac do ban than VK phat trien rat cham (vi du nhu Propionibacterium acnes can 10-12 ngay de phat trien thanh khuan lac tren mdi trudng nudi ca}) [2], [3], [5], [6], [7]. Khi dd, dieu trj se chu }'eu dua vao kinh nghiem, dira phan nao tren ket qua nhudm soi va sir dung cac khang sinh phd rdng, it khang thude. Tren lam sang chimg tdi thudng su dung vancom}'cine 500mg, khi soi tuoi cho ket qua la cau khuan Gr(+), ceftazidime (Fortum Ig) cho true khuan Gr (-) va qumolone the he 3 (Peflacine 400mg x 2 Ig/ngay) cho tap khuan.
Tu} nhien. qua nghien cim nay ngay ca nhudm soi ciing cd the cho ket qua am tinh gia (8/23 - 34.8%) va ca "duong tinh gia"" (7/10 TH nhudm soi cho
tha} ed nhieu loai VK, nhung PCR ket luan lai chi cho tha}' 1 loai). Trong nhirng TH nhu va}. dieu tri hoan toan chi dua vao kinh nghiem lam sang, mang tinh chat la dieu tri thir, bao vay.
PCR va giai trinh tu khac phuc dugc hau het eac nhugc diem cua cae xet nghiem vi sinh kinh dien: (1) lugng benh pham can it han, (2) do nhay xk cao (100% trong nghien ciru nay, 90 - 100%o theo y van); dd dac hieu cao (cd the dat 100% theo y van), (3) tac dung tdt ngay ca khi da dimg khang sinh do kT thuat xac dinh DNA cua VK khdng can VK cdn sdng nhu nudi cay, (4) tra ldi ket qua nhanh trong vdng 24 gid [8], [9], [10]. Trong nghien ciru nay, chiing tdi dat ti le duong tinh
100%), trong dd gan Vi cac TH cho ket qua la phe cau (S. pneumoniae) mdt loai VK cu tni thudng xuyen va gay benh d viing hau hgng. Ngudi Viet Nam cd ti le viem mui hgng rat cao, tuy phan ldn la do virus nhung do dieu tri khdng dimg each, su dung khang sinh bira bai thudng dan den tinh trang benh keo dai, cd the bdi nhiem VK va VK thi cd nhieu nguy co khang thude.
Do dac hieu cua PCR va giai trinh tu phu thude rat nhieu vao do ""tinh khiet cua benh pham"":
cd nghTa la neu benh pham bi nhiem cac chung tap khuan tir mdi trudng ben ngoai se dan den lam sai lac toan bd ket qua [3], [6], [7]. [8]. Qm trinh lay
NGHIEN CUtJ KHOA HOC
benh pham cua chimg tdi rat nghiem ngat, trong dieu kien vd khuan tuyet doi cua phdng md, benh pham duge bao quan trong cac thiet bi vd khuan va d dieu kien vd triing -20° da tao nen do tin cay eua ket qua. Hon nira tat ca qua trinh chay PCR deu cd chimg am va duong de kiem soat nghiem ngat do chinh xac cua phan img.
Nhugc diem cua PCR va giai trinh tu so vdi NCVK la khdng cho dugc ket qua khang sinh dd, ddi hdi trang thiet bi. ma} mdc va gia thanh cao.
Tuy nhien, vdi ket qua dinh danh chinh xac da cd the giup dinh hudng r5 rang cho dieu tri tren lam sang. Han niia, ket qua xac dinh can nguyen gay benh se cd vai trd rat ldn trong nghien ciru ve khia canh dich te hgc lam sang cua benh li VMNNNS do VK. Nhu da ndi d tren, benh VMNNNSVK d Viet Nam ed dac thii rieng so vdi y van: neu nhu cac bao cao d cac nudc phat trien cho thay benh thudng xuat hien d nhirng BN suy giam mien dich, sire de khang (do nhieu nguyen nhan, mac phai hoac bam sinh) [8], thi d Viet Nam benh lai thudng xuyen gap o nhirng BN hoan toan khde manh, khdng cd tien sir benh toan than ro rang. Khi tien hanh nghien cuu, chiing tdi cd gang xac dinh d nhiem tning nguyen phat bang cac kham nghiem toan than hang loat nhu: cay mau, cay nudc tieu, chup XQ phdi, sieu am o bung tdng quat,...
T A I LIEU THAM K H A O
1 BROSIUS, J. M L PALMER, P J KEN- NEDY and H F N O L L E R , 1978: "Complete nu- cleotide sequence of a 16S ribosomal UNA gene from Escherichia coli", Proc. Natl. Acad. Sci.
USA, 754801-4805.
2. N M CAROLE, E E M JAEGER, S C H O U D H U R Y , A A S DUNLOP, M. M. MATHE- SON. P ADAMSON. N OKHRAVI, S LIGHTMAN:
"Detection of and Discrimination between Gram - Positive and Gram - Negatn e Bacteria in Intraocular
Nhung cho den na}. chimg tdi chua xac dinh dugc mdi lien quan nao ro ret. Ket qua tren nhdm nghien cim na.}' cho tha} gan Vi TH tac nhan ga} benh la phe cau, da md ra mdt hudng di mdi cho nghien cim: tim kiem d nhiem trimg nguyen phat d dudng hd hap tren, noi eu tni thudng xuyen cua phe cau.
Cac BN trong nghien cim tiep theo can dugc ngoa}
hgng hang loat de chan doan, khang dinh mdi lien he cd the eua phe cau hgng, hau vdi VMNNNSVK.
Khi lat lai hd so luu trii cua nhiing BN, sau nay xac dinh tae nhan gay benh la do phe cau, that bat ngd la chimg tdi thay benh canh lam sang cd rat nhieu diem tuong ddng: benh xay ra rat cap tinh, ban dau thudng bieu hien nang d ban phan tnrdc vdi cac tdn hai nang eua giac mac (phu giac mac du ddi, ap xe vdng, xuat tiet day d tien phdng) va chinh vi vay thudng de lai di chimg nang ne (loan dudng giac mac) mac dii nhiem trimg da duge dieu tri thanh cdng.
V. KET LUAN
PCR va giai trinh tu cd dd nhay va do dac hieu cao, tra ket qua nhanh ed the giiip ich rat nhieu cho cdng tac chan doan va dieu tri benh VNNNSVK. Han the nira, trong hoan canh Viet Nam, xet nghiem nay cdn hira hen giiip ich rat nhieu cho viec xac dinh ca che benh sinh cua benh, mdt van de ma cho den nay van la mgt ton dgng nhiic nhdi cua nganh Nhan khoa.
Samples by Using Nested PCR", J. Cim. Microtia, May 2000, p. 1753-1757.
3. P GOLDSCHMIDT, S DEGORGE, D BENALLAOUA, et al: "New test for the diagno- sis of bacterial endophthalmitis", Br J Ophthalmol, 2009, 93:1089-1095 originally published online Febmary 10, 2009.
4. H O A N G THI HIEN (2005), "Nghien cuu dac diem lam sang va mgt so tac nhan gay viem mu ndi nhan ndi sinh", Ludn vdn thac sT, Dai hgc Y Ha Ndi.
5. F T KERKHOFF, A VAN DER ZEE, A M C BERGMANS. A ROTHOVA: "Polymerase Cham Reaction Detection of Neisseria meningitidis in the Intraocular Fluid of a Patient with Endogenous Endophthahnitis but without Associated Meningitis".
Ophthalmology, 2003; 110:2134-2136.
6. NOKHRAVI,PADAMSON,SLIGHTMAN:
"Use of PCR in endophthalmitis". Ocular Immunology and Inflammation, 2000, Vol. 8, No. 3, pp. 189-200.
7.N OKHRAVI, P ADAMSON, M M MATHESON, H. M. A. TOWLER, S LIGHTMAN.
PCR-RFLP: "Mediated Detection and Speciation of Bacterial Species Causing Endophthalmitis", In- vest Ophthalmol Vis Sc, 2000, 41:1438-1447.
8. D SEAL. U PLEYER. Ocular infection, 2"<^
ed. Infomia Healthcare USA. hic. 2007: 61-79.
9. DSEAL.UREISCHL.ABEHR.CFERRER.
J ALIO , R J. KOERNER, P BARRY: "The ESCRS Endophthalmitis Study Group. Laborator} diagnosis of endophthalmitis: Comparison of microbiolog}' and molecular methods in the European Societ} of Cataract & Refractive Surgeons multicenter stud}' and susceptibilit}' testing". J Cataract Refract Surg 2008;34:1439-1450.
10. K L THERESE. A R ANAND AND H N MADHAVAN: ""Pol}'merase chain reaction in the diagnosis of bacterial endophthalmitis"". Br J Ophthalmol, 1998 82: 1078-1082.
SUMMARY
USING PCR AND SEQUENCING IN CAUSATIVE DIAGNOSIS OF BATERIAL ENDOGENOUS ENDOPHTHALMITIS
Objectives: Report the preliminary result of PCR and sequencing in bacterial investigations of vitreous samples from suspected bacterial endogenous endophthalmitis patients.
Methods: Non-control descriptive study. Using PCR and sequencing to analyze 23 vitreous samples from 23 clinically diagnosed bacterial endophthalmitis cases (treated in VNIOfrom 4-2008 to 11 2008) in
comparing with conventional microbiological techniques (Gram stain and culture). The tests were done in Biology Department, Gifii university, Japan in Januaiy 2009.
Results: Gram stain showed: 8 23 (34.8%) cases were negative while PCR and sequencing were positive in all cases. In 10 23 (43.5%) cases more than 1 bacteria were seen on Gram stain but only 3 of
them were confirmed on PCR and sequencing. In the remaining 7, only 1 bacteria was detected. Culture showed: low positive rate (3,23 - 13%) but matched with the result of PCR and sequencing. PCR and sequencing showed: nearly 'S (11'23) cases were Streptococcus pneumoniae. The other 5 were mix (e.g.
more than 1 bacteria) without cloning done due to shortage of time and budget. The remaining included:
Enterococcus casseliflanis (1), Streptococcus sp (1), Staphylocuccus sp (1), S. heamolyticus (1), Bacillus sp (1), S. sciuri (1), Enterobacter hormaecher (1).
Conclusion: PCR and sequencing gave much better result than Gram stain and culture did. which would play a vital role in the disease management and also in the idendflcation of the primary infection of the bacterial endogenous endophthalmitis.
Key words: Endogenous endophthalmitis. PCR. Sequencing.
