GIA TR| U'NG DUNG CUA KY THUAT PCR DA MOI PHAT HIEN CAC VI KHUAN HAEMOPHILUS INFLUENZAE, STREPTOCOCCUS PNEUMONIAE VA NEISSERIA MENINGITIDIS TRLFC TIEP Tl> DjCH NAO TUY
Phan Le Thanh Huong, Dang Thu Hang*. Pang Thj Nga, Nguyen \ an Cuong, Nguyen Iran Hien
I 'ifn I "e sinh Dich te Trung trong, lid npi Hfnh vien Nhi Trung uong, Hd npi TOM TAT
Muc tieu ciia nghien ciru la danh gia gia tri ting dung cua ky thuat PCR da mdi trong chin doan can nguyen vi khuan gay viem mang nao mil (VMNM) 61 tre em tryc tiep tir djch nao tuy Phuong phap PCR da moi
phat hien dong thdi 3 tac nhan vi khuan chii yeu gay VMNM a tre nhd la Haemophilus influenzae cd vd (trong dd cd Hib), Streptococcus pneumoniae va Neisseria meningitidis da dirge xay dung va ap dung de xet nghiem cac mlu benh phim dich nao tuy (DNT) ciia benh nhi diro'c chin doan xac dinh viem mang
nao do vi khuln (dua tren trieu chirng lam sang va sir bien ddi te bao, sinh hoa ciia dich nac tuy) Cac chi sd dd nhay. dp dac hieu, gia trj du doan ciia phan irng PCR da moi da duoc tinh toan nhlm do ludng tinh gia tri ciia ky thuat Xet nghiem tren 231 mlu DNT (trong dd cd 131 mlu tir benh nhi du'oc chin doan xac djnh viem mang nao do vi khuln va 100 mlu tir benh nhi viem nao do virus), ty le phat hien dupe vi khuan bdi PCR da mdi so vdi ty le phat hien ciia nudi cly phan lap cao hon tir 1.5 (vdi Hib) den 3.1 lan (vdi S pneumoniae va N. meningitidis) va cd y nghia thing ke (P < 0,05). Dp nhay ciia PCR da moi vdi Hib la 90,9%; 100% vdi S pneumoniae va 100% vd-i N. meningitidis. Dp dac hieu ciia PCR da mdi vdi Hib, S, pneumoniae va N. meningitidis deu dat 100% Gia tri dir doan am tinh khi kit qua xet nghiem PCR da mdi am tinh doi vdi Hib la 97,1%; 100% vdi S. pneumoniae va 100% vdi N. meningitidis. Ky thuat PCR da moi nen duoc si> dung trong chin doan bdi ky thuat nay dupe coi la mot giai phap nhlm nang cao chit luong xet nghiem phat hien tac nhan gay benh, khlc phuc cac han chi cua ky thuat kinh dien, gdp phln nang cao hieu qua dieu tra
Tir khoa: PCR da moi, Streptococcus pneumoniae. Neisseria meningitidis Hib, viem mang nao mil I. DAT VAN DI
I rony dieu kien kinh te - y te ciia Vie!
Nam hien nay, vice chan doan tac nhan vi sinh g;i\ benh nhiem triing he than kinh trung uong ndi chung va dac biet d Ire nhd van cdn rat nhieu han che. gay anh huong tdi hieu qua dieu trj va chi phi y tc. Haemophilus influenzae cd vo (chu yen la I lib). Streptococcus pneumo- niae \a Neisseria meningitidis la nhung vi
khuan tlurdn g gay viem mang nao mu. elite biet d tie nhd. Tai Viet nam. cho den nay chan doan phong thi nghiem van chu yen dua vao nudi cay phan lap vi khuan tir dich nao Hi>.
Phirong phap nudi cay phan lap vi khuan cho ket qua chinh xac nhung ty le duong tinh thap va thdi gian lau. Phuong phap phan irng chudi men poly nlcrasa ( Polymerase chain reaction PCR). dac biet PCR da mdi la phuong phap I in tac gia: Phan Le Thanh Huong
Dia chi: Khoa Vi khuan. \ ien Ve sinh Djch tc Trung uong. sd I Yersin. Ha Ndi Dien thoai: (04) 3971 4408
Lmail: thanhhuong'oJnihe.org.vn
Tap chi Y hoc dir phong. Tap XX. so 6 i I 141
phat hien va khuyech dai cung mot luc cac doan gicn dac hieu cua cac loai vi sinh vat khong can vi sinh vat song, khac phuc duoc tinh trang v i khuan gay benh da bi ire che hay bi diet boi khang sinh. Hon the PCR con cho ket qua nhanh trong mot ngay. De tang hieu qua trong chan doan va giam sat dich te hoc ciing nhu co cong cu danh gia chinh xac ganh nang benh tat do nhirng v i khuan nay gay ra.
chiing toi ap dung qui trinh PCR da moi da duoc hoan thien trong nghien ciru trudc (tren mo hinh) doi voi cac man djch nao tuy (DNT) thu duoc tir cac benh nhan da duoc chan doan lam sang viem mang nao do vi khuan nham danh giii cac gia tri do nhay, do dac hieu. gia trj dir bao am linh... cua phuong phap PCR da moi khi so vdi cac phuong phap chuan thirc khac. tir dd de xual phuong phap hieu qua.
II. rTIUONG IMlAP NGHIEN ( IT 2.1. Cluing thuan:
Ilarmophilus influenzae iyp b 'III1- AICC 35056): Neisseria meningitidis (AK'C 13090): Streptococcus pneumoniae (ATCC (i303())duac su dung lam cluing duong. Nhung cluing vi khuan khac dudi day la nhung cluing gay benh plijin lap tu benh pham duoc su dung de xac djnh tinh-dac hieu ciia PCR da mdi:
//. influenzae khdng co vu. V gonorrhoeae.
Streptococcus suis. Streptococcus mitis, Stap- hylococcus aureus. Enterococcus faecalis.
I: coli. Klebsiella pneumoniae va bach cim ngudi. I iich chict ADN cua \ i khuan bang bo sinh pham cua QlAgcn \ a bang phuong phap nhiet do (100'C 1 ()'). Giu ADN o -2()"C.
2.2. Benh pham dich nao tin, nudi cay phan lap v ;i bao quan:
131 mau dich nao tuy (DNT) tir cac benh nhan duoc chan doan viem mang nao do vi khuan dua tren trieu cluing lam sang va cac chi so le bao \ ; i sinh hda DNT (bach can da nhan trung tinh > 100 le bao ml: glucose giam
< Tiling 01: protein > 45mg dl); 100 mau DNT
tir benh nhan duoc chan doan xac djnh viem nao do vi riit (xet nghiem \ icm nao Nhat ban 13 | 11) hoac chi la hoi cluing nao cap (cite bieu hien t i bao \a sinh hda DNT gan nhu binh thudng. xet nghiem v i sinh am tinh). Cac mau DN I cua benh nhan dupe nudi cay phan lap theo thudng qui cua CDC: ngay dau tien city vao canh thang lang sinh BI II cd bd xung 5%
levinthal (cluia cac yeu to dinh dudng ke ca X va V). ngiiy thu hai cay ra thach sdcdla va thach mau ciru 5%. ii o 37"C ?°.nCO, trong 24 gid. Vice djnh danh vi khuan hoac djnh typ huy et thanh dua \ ao vice xiic dinh cac dac tinh tirng loai vi khuan \ ; i khang huyet thanh dac hieu duoc thuc hien vao ngay tiep theo neu xuat hien cac dang khuan lac nghi ngd tren mdi trudng thach man (ddi \ di .V pneumoniae va V. meningitidis) hoac tren thach sdcdla (vi khuan Hib). Viec nudi c;i\ vi khuan dupe thuc hien tai Khoa Xet nghiem Vi sinh ciia Benh vien Nhi Trung uong. Benh pham DN T dupe bao quan trong cac dng cryotube mil xofty ngoai. gift o - 80 (
2.3. Vat lieu cho PCR da moi:
3 cap gen mdi (thudc viing I6S r.ARN) dac hieu luong irng voi Hib. S. pneumoniae va A meningitidis cd trinh tir lan luri,fl la: voi Hib: 111.I: 5"-CG 1 TIC, TAI GAI G I I < -.\.
CCA GAC T-3'; III.2: 5 - TGT CCA TG I t i l l CAA A AT GAT G-3 .Kich thudc san pham: 3 13 bp. Vdi V pneumoniae l.ytA. T 5'-CGG AC T ACC Gt'C T'TT ATA TCG-3";
l.yl.A. R: 5'-GTT I CA ATC GTC AAG CCG TT-3'. Kich thudc san pham: 229 bp. Vdi A meningitidis CtrAllOF: 5'- OCT GCG GIA GOT GG'T TCA A-3"; CtrAllOR: 5"-TTG
ICG CGG ATT TGC A AC TA-3". Kich thudc san pham PCR: 110 bp; Tac/ polymerase (in- vitrdgen Co.. Singapore); dNTPs (Invitrogen Co.. Singapore) 400 uM: MgCI, 25 mM; dung djch Triton X-100 (Merck): nude tinh sach (Sartorius); thach Seakem G'TG Agarose".
thach 'NuSic\c GIG Agarose' (C'ambrcx Bio Tap chi V hoc du phong. Tap XX. so 6(114) 29
Science Rockland. Inc. USA): TAI lOx (Pro- mega Corporation I SA): ethidiumbromide;
loading buffer (No\agen Co.): PCR Markers 100 (Novagcn Co.). Bo ngirng ket latex phat hien khang nguyen vo dac hieu ciia cac loai vi khuan dich Wellcogen Bacterial Antigen Kit (ciia Remel Europe Ltd Clipper Boulevard West Crossways. Dartford. Kent 1)A2 6PT. UK).
2.4. Phuong phap phat hien khang nguyen bang phan ling ngu rig ket latex (NKLA):
Duoc thuc hien theo thudng qui cua nha san xuat vdi cac man dich nao lily ciia benh nhan vdi the tich 40 u4 DNT. tai Phong thi nghiem Vi khuan hd hap. khoa Vi khuan.
Vien Ve sinh Dich tc trung uong.
2.5. \ u ly djch nao tuy:
Co 4 phuong phap xu ly DNT da duoc so sitnh vdi nhau va vdi phuong phap xu ly bang bd tach chiet ADN thuong phiim QIAgen.
Phuong phap tdi uu da duoc danh gia trong nghien ciiu trudc nhu sau: mdi man DNT ciia benh nhan duoc lay 500ul vao dug eppendorf, ly lam 15.000 vdng 10 phut, bd nude ndi. gid lai 100 u.1 phan lang. trdn deu vdi 10 pi Triton X -100. each thuy 100"(71 Ophiit. lam lanh va gifr o -2()"C nhu la ADN khudn.
2.6. Thuc hien PCR da nidi:
Sau khi can chinh, ham luong toi uu ciia cac thanh phan cua 1 phan ling PCR da nidi nhu sau: ham luong mdi dung trong phan ling la 20 pmol mdi loai; ham luong Tag polyme- rase 1,5 don \ i phan irng: ndng do dNTPs 200 uM. ndng do MgC'l 2.5 mM. 5 pi dem PCR (duoc cung cap \ di Tac/) 5x: nude tinh sach bd sung \ ua du 25j.il: \ a 1-2 pi ciia mdi man dich nao tuy da xu ly. Phan ling dupe thuc hien o
qui H inh PCR da mdi cd 2 chu trinh nhiet: chu trinh thii nhal cd 2 chu ky ciia 96"C x .V;
55"C x 3()"va 72"C x 30". Chu trinh thii hai cd 35 chu ky vdi 94 C xl':55"C x U: 72llC x 1'; thdi gian keo dai cudi ciing d 72"C la 8 phut tai may PCR Mastercy cler epgradient S (Eppendorf)
2.7. Dien di phat hien san pham PCR:
Su dung hon hop 2 thach dien di "Sca- kem GTG Agarose' \a NuSievc GTG Agaro- se', pha irong TAli ty le 1:1. the tich san phiim PCR chay dien di la 10 pi. Sau dd nhuom gel vdi 1% ethidium bromide vii dpc dudi anh sang UV (BioDoc-ll. Imaging System - LA P. USA) 2.8. Xir ly so lieu:
Vi ket qua nudi cay phan lap ludn bi han che. khdng phan anh diing so lieu duong tinh va am tinh thuc bdi vice sir dung khang sinh trudc chan doan rat phd bien. nen de tinh cac chi sd danh gia gia tri cua phuong phap PCR da nidi khi ap dung trong thuc tc. cluing tdi phai dua tren vice so sanh ket qua phai hien dupe vi khuan bdi PCR da mdi vdi ket qua phat hien vi khuan bdi phuong phap nudi cay phan lap va phat hien killing nguyen (ngung ket latex). Nguyen tac tinh nhu sau:
Sir dung tat ca cac man dupe thuc hien ddng thdi 3 phuong phap (nudi cay. latex.
PCR da mdi) va coi ket qua duong linh d nipt trong hai phuong phap nudi cay va latex dupe coi la mini (+) thuc. Cac mau am tinh thuc khi ca hai phuong phap nudi cay va latex deu am tinh (mini duong tinh trong PCR da mdi khdng dupe tinh vao cac man duong tinh thuc bdi PCR da mdi dang lii phuong phap dupe danh giii).
'Tap chi Y hpc dir phong. Tap XX. so 6 (1 141
Bang s6 lieu co ban de tinh toan cac chi s6 Phuong phap da cong bo
Duong tinh Am tinh Tdng
Duong tinh Phuong phap thu nghiem
Am tinh
a b c d
a+b c+d
Tdng a+c b+d a+b+c+d
Do nhay hay ty le duong tinh that a (a-c) Do dac hieu liay ty Ic am tinh that d .(b-d) Ciia tri du doan am tinh (NPV) d' (c+d)
Kiem dinh Khi binh phuong (/'- test) hoac Fisher exact test duoc thuc hien de so sanh su khac biet gifra hai ty le.
III. KI T QUA
3.1. Ket qua xac djnh duoc vi khuan bdi PCR da mdi (Bang I va Hinh I) Bang 1. Ket qua phat hien 3 loai vi khuan bang PCR da mdi
Vi khuan (n = 131) Hib S. pneumoniae N. meningitidis
S6 mlu (+) 56 (42,7%) 28 (21,4%) 6 (4,6%)
S6 mau (-) 75 (57.3%) 103 (78,6%) 125 (95,4)
Trong 131 mau DN I lay tir 131 benh phap PCR da mdi cd 42.7% (56 13 I) duoc xac nhi cd chan doan xac djnh viem mang nao do djnh do vi khuan 1 lib. 21.4% (28/13 1) duoc xac
\i khuan dua tren trieu cluing lam sang va xet djnh do \ i khuan .V. pneumoniae va 4,6% (6. 13 1) nghiem sinh hda tc bao DNT. Bang phuong duoc xac djnh do v i khuan A. meningitidis.
Hinh 1. Ket qua phat hien vi khuan gay benh trong DNT bang PCR da moi
/ \l: Thang chuan IDA; (7 ' I: chiing duong ca 3 loai vi khuan trong I dng phan irng; C(-):
cluing dm; ST-S/-I: matt benh pham DXT.
Tap chi Y hoc du phong. Tap XX, so 6 (114) 31
1 linh 1 cho tha\ cung mot liic, neu benh pham chua 1 trong 3 loai vi khuan dich co the duoc phat hien trong cung mot thai gian phan ung cua PCR da moi (thay vi de phat hien moi loai vi khuan phai chay mot loai phan ting):
benh pham so 2.6 va 13 duong tinh voi llib.
benh pham so 7 \ a 11 duong tinh \ di .V. pneu- moniae: benh pham so 9 (S9) va so 14 (S14) duong tinh vai A', meningitidis.
3. 2. Danh gia do nhay, do dac hieu va gia trj dir nao am tinh ciia PCR da moi:
Bang 2. Cac chi s6 danh gia tinh gia trj ciia phirong phap PCR da m6i Chi s6 danh gia HIB S. pneumoniae N. meningitidis
Donhay(%) 90,9 100 100 Do dac hieu (%) 100 100 100 Gia tri du doan am tinh (%) 97,1 100,0 100
Hang tren cho thay ket qua phat hien cua PCR da moi khi so sanh voi 2 phuong phap nuoi c;i> phan lap (NCPI.) va ngung ket latex (NKI.A): do nhay cua phuong phap PCR da moi phat hien vi khuan Hib dat gan 91% (90.9); Trang trudng hop xet nghiem am tinh vdi vi khuan Hib. xac xuat de benh nhan khdng mac benh do llib la 97.1%: vdi 2 loai
vi khuan S. pneumoniae va A. meningitidis dd nhay deu dat 100%. Trong trudng hop xet nghiem am tinh vdi vi khuan S. pneumoniae.
xac xuat de benh nhan khdng mac benh do vi khuan na\ la 100% va xac xuat khdng mac benh khi ket qua PCR am tinh vdi A. menin- gitidis ciing la 100%. Do dac hieu ddi vdi ca 3
loai vi khuan deu dat 100%.
3.3. So sanh gia tri phat hien vi khuan cua 3 phuong phap:
Bang 3. Ket qua phat hien 3 loai vi khuln bdi PCR da moi, nudi cay phan lap va phat hien khang nguyen
Tong so DNT viem nao do vi khuan = 131
Phirong phap HIB S. pneumoniae N. meningitidis
Nuoi cay 38 (29%) 09 (6,9%) 02 (1,5%)
Ngung ket LA 52 (39,7%) 18 (13,7%) 03 (2,3%)
PCR da mdi 56 (42,7%) 28 (21.4) 06 (4,6%)
Tap chi Y hoc dir phong. Tap XX. so 6 (I 141
I v Ic phat hien duoc v i khuan Hib trong tong so cac mau benh pham djch nao tuy cua benh nhan duoc chan doan viem mang nao do vi khuan (n 131) bang phuong phap nuoi cay phan lap la 29%. bang phuong phap xac dinh khang nguyen la 39.7% va bang phuong phap PCR da moi la 42.7%. Tuong tir nhu vay
voi S. pneumoniae lan lupt la 6.9%; 13,7% \a 21,4%. voi A. meningitidis lan luxrt la 1.5" o;
2.3% \a 4.6%. Nhu \ay. ty le phat hien bang phuong phap nuoi c;iy phan lap dat thap nhat trong 3 phuong phap va ty le phat hien bang phuong phap PCR da moi dat cao nhat.
Bang 4. Gia trj dir doan am tinh cua ba phircmg phap phat hien vi khuan Phirong phap Vi khuan HIB'" Phd cau|b| Nao mo cau|e|
Nuoi cay
Kit thuong pham NKLA PCR da m6i
75.6 % 90.4 % 97.1 %
85.1 % 92.5 % 100%
96.9 % 100%
100%
Doi vdi vi khuan I lib; gia trj dir doan am tinh ciia phuong phap nudi cay lhap hon hai phuong phap latex va PCR da mdi (/' < 0.05).
khdng cd su khac biet cd y nghTa thdng ke gifra phuong phap latex va PCR da mdi (P — 0.515).
Ddi vol S. pneumoniae: phuong phap PCR da mdi cd gia tri dir doan am linh cao nhat (100%) va cao hon hai phirong phap con lai (/' < 0.05). khdng cd su khac biet cd y nghia thdng ke gifra phuong phap ngung ket latex \a PCR da mdi.
Ddi vdi A meningitidis : ca ba phuong phap deu cd gia trj du doan am tinh tuong duong nhau.
IV HAN M A N
Ap dung tren 131 mau benh phiim lam sang (duoc chan doan xac djnh \ iem mang nao do \ i khuan boi cac tieu chuan chan doan lam sang va sinh hda te bao djch nao tuy). phirong phap PC R da mdi da cho kel qua xac djnh vi khuan ca 3 loai cao hon han nudi cay phan lap lir 1.5 lan (ddi vdi llib) den 3.1 lan (ddi
viii .9. pneumoniae va A', meningitidis) va sir khac nhau nay cd y nghTa thdng ke (P < 0.05).
nhung khdng cd sir khac biet cd y nghTa khi so sanh kha nang phat hien ciia PCR da mdi vdi phuong phap phat hien khang nguyen (NKLA;
P > 0.05). Dieu nay cd y nghTa rat quan trong d Viet Nam bdi vi viec lam dung khang sinh dang rat phd bien, gay anh huong Idn tdi ket qua chan doan tac nhan vi sinh vat gay benh bang phuong phap nudi cay phan lap. Hon nCia. PCR da mdi con cd mot uu the la nhanh.
cho ket qua chi trong vdng 3-4 gid trong khi nudi cay phan lap can it nhat 48 gid. Ngoai ra. PCR da mdi con cd the phat hien dupe nhirng trudng hop da nhiem. cu the la trong
131 man djch nao tuy cd 2 man DN T (1.5%) da ddng thdi phat hien dupe 2 loai vi khuan (5. pneumoniae va Hib). trong dd cd mot loai vi khuan d ndng dp thap (so lieu khdng dupe trinh bay).
Dp nhay. dp dac hieu va cac gia trj dir doan ciia PCR da mdi dupe tinh dua tren vice so sanh voi ket qua phat hien ciia nudi Tap chi Y hoc du phong. "Tap XX. so 6 (1141
cay phan lap- chuan vang nhung bi han che do viec lam dung khang sinh d Viet Nam va ket qua phat hien khang nguyen (khong can \ i khuan song) da cho ket qua: do nhay ciia PCR da moi doi voi ca 3 loai vi khuan deu dat tren 90% (90.9% vai Hib; 100% voi .V pneumoni- ae va .V. meningitidis): neu tinh theo so luong v i khuan moi loai ma PCR da moi co the phat hien duoc la I vi khuan/phan ting. Do dac hieu ciia PCR da moi vai ca 3 loai vi khuan deu dat do chinh xac cao (100° u). Xac xuat khong mac benh do tirng loai \ i khuan dich thong qua gia trj phat hien am tinh ciia PCR da moi doi voi Hib la 97.1% va 100% ddi voi 2 loai vi khuan con lai. |Bang 41.
I iiy nhien. o nghien ciiu nay. trong vice phat hien \ meningitidis (nao mo can), ca hai phuong phap sir dung lx.it thuong pham va PCR da moi co do nhay ngang nhau va cao gap 3 lan phuong phap nuoi cay. Tuy viiy. su khac biet gifra ba phuong phap nay khong co y nghTa thong ke (P > 0.05). co the do so trudng hop nudi cay duong tinh cldi vdi nao md ciiu qua it. nhirng ty le phat hien duoc nao md can so vdi tudi trong nghien ciru nay la hop ly vi dai da so benh pham DNT thu duoc a day deu thudc nhdm tre dudi 5 tudi (khdng phai nhdm ddi tuong chinh cua viem mang nao do nao md can (A meningitidis).
Gia trj dir doan am tinh tang dan tir phuong phap nudi cay. NKL A va cao nhat d PCR da mdi khi ket qua PCR am tinh. Ddi vdi Hib tir 75.6 den 97.1%, vdi S. pneumoniae til 85.1 den 100% \;i vdi A meningitidis tir 96,9 den 100% (Bang 3.4). Sir khac nhau ve gia trj du doan am tinh giua phuong phap PCR da nidi \ ii nudi cay phan lap cd y nghTa thdng ke, dac biet ddi llib va S. pneumoniae (/•<0,05), nhung khdng khac nhau cd y nghTa gifra PCR.
da mdi va NKLA (/>>0.05).
\. KI T I I \\
Ky thuat PCR da mdi cd the- cho ket qua
nhanh (trong ciing ngay thu benh phiim). do nhay dat > 90%. do dac hieu dai 100%.. tier kiem sinh pham iV_ cdng sire (ciing mot dug phan irng cd the phat hien duoc 1 den 3 loai \ i khuan chii yeu giiy benh). Ket qua cua PCR da mdi cd gia trj dir doan am tinh cao hon nudi cay phan lap (P< 0.05). Ky thuat PCR da mdi dirge coi la mot giai phap nham nimg cao chat luong xet nghiem phat hien tac nhan giiy benh.
khac phuc cac han che cua ky thuat kinh dien (NCPI.). gdp phan nang cao hieu qua dieu tri.
L&i cam on: chung tdi xin chdn thanh cam on sir tat tro kinh phi cua C Turong trinh Tiem chung Mo' rang Oudc gia. chdn thanh cam on Pan Giam doc yd Khoa Xet nghiem \ i smh cua Benh vien Xin '/'rung uong dd ddng y vd lao dieu kien giup chiing idi cd diroc sd luong benh pham can thiet thirc hien trong nghien ciru. Xin chdn thanh cam on Vs. Tran Quang Binh dd giup xir ly. linh loan vd phan tich cue di~r lieu nghien cuu.
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VALIDITY OF MULTIPLEX PCR ASSAY FOR DIRECT DETECTION OF HAEMOPHILUS INFLUENZAE, STREPTOCOCCUS PNEUMONIAE AND NEISSERIA MENINGITIDIS IN CEREBROSPINAL FLUID
Phan Le Thanh Huong. Dang Thu Hang". Tang Thi Nga. Nguyen Iran 11 ien
\titional Institute of Hygiene and Epidemiology, Hanoi
* National Hospital of Pediatries, Hanoi The Objectiv e of this study is to assess
the applicable valid ity of a multiplex PCR tech- nique to detect directly meningitis bacteria. A multiplex PCR technique was developed and applied to detect Haemophilus influenzae (in- cluding Hib). Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid (GST) of pediatric patients diagnosed based on clinical sy mptoms and abnormal changes of cellular & biochemical indexes in CST).
The sensitiv ity, specificity and predictive val- ues of the multiplcx-PCR technique were cal- culated. A results of testing 231 CSP samples (131 CSl's from confirmed bacterial meningi- tis cases and 100 CSLs from confirmed v iral encephalitis cases) showed that the detective rates of the multiplex PCR were higher those of culture method from 1.5 (for Hib) to 3
times (for S. pneumoniae & ,V. meningitidis) and significantly (P<0.05). The sensitivity of the multiplex PCR method was 91% to Hib:
100% to S. pneumoniae: 100% to A. menin- gitidis. The specificity of the multiplcx-PCR to all 3 bacteria was 100%. The negatively predictive value when getting negative result in multiplex PCR to Hib was 97.1%; 100% to -S". pneumoniae and 100% to A. meningitidis.
fhe difference of bacterial detection between multiplcx-PCR and culture method was sig- nificant at P< 0.05. The multiplcx-PCR tech- nique should be used to improve the testing of meningitis pathogens, overcome limitations of the classic method and improve treatment effectiveness.
Key words: bactccial meningitis, mul- tiplex. PCR strcpocouas
Tap chi Y hoc du phong. Tap XX. so 6 (1 141 35
