DANH GIA HIEU QUA PHAT HIEN TRlTNG GIUN SAN
B A N G
PHirONG PHAP ETHER-FORMALIN CAI TIEN
Nguyen Thu Hirong, Nguyen Thi Viet Hoa, Le Xuan Hung
Tom tdt
Ky thuat ly tam ldng can ether-formalin ldn ddu tien dupc giai thi?u bai Richie nam 1948. Den nay ky thudt ndy vdn dupc lua chpn trong cdc nghien ciru phdt hien giun sdn vd dan bdo trong phdn.
Ky thudt ether-formalin cdi tien trong nghien cihi ndy nhdm tdng khd ndng phdt hien vd thu hdi sd lupng trimg ky sinh triing duang rupt. Kit qud nghien cim cho thdy ky thudt ndy phdt hien trimg giun sdn vd dan bdo tdt hon ether-formalin cita Suzuki ndm 1981. Ky thudt ether-formalin cdi tien dd thay bdng Ipc qua mpt lap gac, khong rira vd chi ly tdm 2 ldn bdng Ipc qua 3 lap gac, rira lgi 3 ldn vd them mdt buac ly tdm Ipc rira lgi ldn thd ba. Ddy la mpt ky thudt cd the dp dung trong nhitng nghien cim ve hieu qud dieu trf, ddnh gid khdng thudc giun sdn. Nd hiea hen dp dung tdt tgi cdc diem dd cd chuang trinh phdng chdng giun sdn nhieu ndm, nai md ty I? nhiem vd cuong dp nhiem dd gidm.
1. DAT VAN DE
Cac benh ky sinh triing dang la mdt trong cdc van de y te cdng ddng chinh cua cdc nudc dang phdt tridn[2]. Viet Nam la mdt nudc nhiet ddi cd day dii cdc dieu kien vd mdi trudng, khi hdu, kinh td, xa hdi va tap quan sinh boat thich hop cho sir phdt trien va ldy lan ky sinh trung dudng rupt trong cdng ddng [4]. Trong nhftng nam qua, chuang trinh phdng chdng giun sdn qudc gia dupe sir ho trp cua cac td chiie qudc td va chinh phu da va dang trien khai cd hieu qud trdn toan qudc. Nghien ciiu va ling dung cdc phuang phdp chan dodn giun sdn hien dai nhu huyet thanh chdn doan (ELISA), kfch dai gen bdng phan iing chuoi sinh hpc phan tu (PCR) rdt cdn thidt nhdm chdn dodn sdm va didu tri dae hidu benh giun sdn. Tuy nhien, cdc phuong phdp nay khd cd thd thay thd cdc phuang phdp xet nghiem phdn ca bdn. Moi phuang phap xet nghiem phan ddu cd eac uu nhuac didm khde nhau. Cdc loai tning ky sinh triing dudng rupt rat da dang vd hinh thai hpc, chiing dupe phan bidt dua vao cdu tnic vd, mau sdc, hinh dang, kfch thudc va cdu tnic nhdn. Vd tining ed loai xu xi va day, ed loai vo mdng va tirong. Cdc loai triing giun thudng cd dang hinh Oning, hinh bdu due, trdn, tning sdn day thudng cd 2 ldp vd, tning san Id cd ndp. Khd ndng phdt hien tning giun sdn ti-ong phan phu thupc nhidu vdo timg loai ky thudt xet nghiem, vao kinh nghiem cua ngudi ky thudt vidn va ddc bidt vao cudng dp tning trong phan. Ky thudt ly tam ldng can ether-formahn (EF) la ky thudt lan ddu tidn dupe gidi thieu bdi Richie nam 1948[5], ddn nay van la ky thuat dupc lira chpn ti-ong cdc nghien ciiu va chdn dodn phat hien ky sinh triing trong phdn. Ky thudt nay khdng chi ed dp tin cay dd phdt hien tning giun san, du triing va bao nang don bao ma cdn nhidu uu didm nhdt dinh ke ca vide it lam bien dang
Dja chi lien h§:
ThS. Nguyen Thu Huong
Khoa Ky sinh triing - Vien Sot rit - KST - CT TU"
DT: (04) 38543250, 0912085470 E-mail: [email protected]
cdu mic hinh dang tning va lam tdng sir phdt hien. Tuy nhien, khd ndng phdt hien ming giun sdn va bao nang don bao ti-ong phdn eiia ky thudt nay ehi vao khodng 6-49% [5]. Do do ehiing tdi tidn hanh nghien ciiu nay vdi muc dfch so sanh khd ndng phdt hien va tap trung tning giun sdn va don bao cua ky thuat Kato-Katz cua Td chiic y td thd gidi, ether-formalin ciia Suzuki, vdi mpt ky thuat ether-formalin cdi tidn.
2. PHLTONG PHAP NGHIEN CUtJ 2.1. Doi tuffng vd dja diem nghien ctru
Cdc mau phan tuoi thu dupc tir cdng ddng ddn cu tai mdt viing trong mau thudc thdn Ddng Tien, xa Mai Trung, huyen Hiep Hda, tinh Bdc Giang. Tdng sd ed 131 ngudi ddn dia phuang tinh nguyen tham gia nghien ciiu. Trong do cd 49 nam va 82 nii, liia tudi tir 2 tudi den 80 tudi.
2.2. Phuffng phdp vd ky thuat nghien cdu
Cdc mdu phdn dupc lay tir ngpy hdm trudc hoac ciing ngpy thu phan. Phan dupc bao qudn trong 1 lp nhua cd nhan ghi rd hp tdn, ma sd, tudi, gidi, dia chi. Mdi ngudi dupc hudng dan ldy khoang 10 gam phan giira bdi phan. Cdc mau bdnh pham dupc dua vd xet nghiem tai Labo khoa Ky sinh trung, Vidn Sdt ret-KST-CT TU".
Moi mdu phdn dupc trdn ky va lam 4 ky thudt: Kato Katz (WHO, 1994), ether- formaline Clia Suzuki, 1981 va ether-formaline cai tien (EFC) vdi dung dich formaline 10%
dieu chinh dp axit pH 4 va pH 3.
Sau khi xet nghiem phdn, nhiing ngudi nhiem giun sdn se dupc didu tri dac hidu bang thudc tay giun sdn theo phde dd Bd Y te ban hdnh [2].
2.3. Ky thudt Ether-Formalin cdi tien
Can 1 gam phan cho vao tube ly tdm, eho 8 ml dung dich formalin 10% vao tube, trdn ky. Lpc dung dich trdn qua 3 ldp gac. Cho them 5 ml dung dich formalin 10% vao tube (de nia gac). Ly tam tdc dp 3.000 vdng/phiit x 2 phiit. Dd dung dich ben trdn di, cho them 6 ml dung dich formalin 10% vao tube. Cho tiep 2 ml ether vao tube. Ldc manh trdn ddu ether vdi dung dich trong tube. Ly tam tdc dp 3.000 vdng/phiit x 2 phiit. Sau do do dung dich ben tren di, cho them 6 ml dung dich formalin 10% vao tube. Ldc ky. Lpc dung dich qua 3 ldp gac.
Cho them 5 ml dung dich formalin 10% vao tube (dd nia gac). Ly tdm tdc dp 3.000 vdng/phut X 2 phiit, rdi do dung djch ben tren di, ldy can soi phdt hien ky sinh triing bang kinh bidn vi d dp phdng dai xlOO va x400.
2.4. Chi so ddnh gid
Ty Id nhidm ky sinh triing cua tung ky thuat se dupc tinh todn so sanh theo thdng ke y sinh hpc. So sanh sir khde bidt giiia hai ty Id dua vao test x^ va hai gid fri ti-ung binh dua vao test Student (Test-T) vdi khoang tin cdy 95%.
3. KET QUA
Tdng sd 131 mau phan dupe xet nghiem bdng ba phuong phdp khac nhau, ether-
formalin cua Suzuki, 1981, ether- formalin cai tien va Kato Katz (WHO, 1994). Ket qua phdt
hien ciia timg ky thudt duac the hien d Bang 1.
Bang 1: Kdt qua xet nghiem phan theo ba phuong phdp: EFC pH = 3, EF va Kato Katz
Loai KST
Triing giun diia Tning giun tdc Tning giun mdc Tning san la nhd Bao nang don bao
EFC vdi pH = 3
s6 +
11 39 26 28 2
%
8,4 29,8
19,8 21,4 1,5
EF
s6 +
7 31 21 19 0
%
5,3 23,7
16,0 14,5
-
Kato Katz
s6 +
7 26 24 6 0
%
5,3 19,8 18,3 4,6
-
P
>0,05
<0,05 0,05
<0,05
Ty le nhiem tning giun sdn theo 3 ky thudt tuong iing: tning giun diia la 8,4%, 5,3% va 5,3%, tning giun tdc 29,8%, 23,7% va 19,8%; tning giun mde 19,8%, 16,0% va 18,3%; tning san Id nhd 21,4%, 14,5% va 4,6%; don bao 1,5%, 0% va 0%. Ty Id nhiem tning giun tdc va sdn Id nhd bdng 3 phuong phdp khac bidt cd y nghia thdng ke (dp tin cay 95%, p < 0,05).
Rieng vdi ky thudt EFC da phat bidn 2 trudng hpp nhidm bao nang Endolimax nana, trong khi 2 ky thudt kia khdng phdt hien dupc.
Bang 2: Ket qua thu tning giun sdn theo 3 phuong phdp EF, EFC va Kato Katz
Loai KST
Tning giun diia Tning giun tdc Tning giun mdc Tning sdn Id nhd Bao nang dan bao
EFC vdi pH 3 S 6 w
2.227 815 1.934 7.517 10
S o w T B 202,5
20,4 71,6 268,5 5,0
E F So w
2.057 743 1.880 7.579 0
S o w T B 293,9
24,0 89,5 398,9 -
Kato Katz S o w
14 63 762 91 0
S o w T B 7,0 4,8 33,1 15,2 -
Ghi chu: TB: trung binh; w: trimg
Tdng sd cd 131 mau phan da dupc phan tfch bang ba phuang phdp EFC vdi pH=3, EF va Kato Katz. Sd tning giun diia thu dupc tuong iing vdi 3 ky thudt la 2.227, 2.057 va 14 tning; giun tdc 815, 743 va 63 tning; giun mdc la 1.934, 1.880 va 762 tning; san Id nhd la 7.517, 7.579 va 91 tning. EFC vdi pH 3 phdt hien dupc don bao Endolimax nana (10 bao nang) cdn EF va Kato Katz khdng phdt hien dupc.
Bang 3: Ket qud xet nghiem theo phuang phdp EFC vdi dung dich formaline 10% pH bdng 3 va pH bdng 4
Loai KST
Trirng giun dua Tning giun tdc Tning giun mdc Tning sdn Id nhd Bao nang don bdo
Ky thuat xet nghiem EFC pH = 3
s6 +
8 25 26 28 2
%
7,9 24,8 25,7 27,7 2,0
pH = 4
s6 +
5 20 23 16 -
% 5,0 19,8 22,8 15,8 0
P
>0,05
<0,05
<0,05
>0,05
Vdi 101 mdu dupc so sdnh kdt qud EFC vdi dung dich Formalin 10% pH = 3 va pH = 4 cho thay ty Id nhiem tning giun sdn phdt bidn vdi PH = 3 cao hon mdi trudng pH = 4. Ty le nhiem tning giun san theo hai ky thudt tuong iing: tning giun diia la 7,9% va 5,0%, giun tdc la 24,8% va 19,8%, giun mdc la 25,7% va 22,8%, sdn Id nhd la 27,7% va 15,8%, bao nang don bao Endolimax nana la 2,0% va 0%. Sir khde bidt nay chi ed y nghia thdng kd vdi tning giun tdc va giun mdc (dp tin cdy 95%, p < 0,05).
4. BAN LUAN
Ether su dung trong ky thudt EF dung de hda tan md va lam ndi manh phan, lam cho triing vd ken ky sinh triing tdch ra khdi manh phan trong khi ly tam. Kdt qua tdng hpp cua Tankan va CS, 2008 eho thdy vdi phuang phdp EF thi 2% so triing giun sdn bdm tren be mat thanh tube, 27% d ldp gac lpc, va 36% tning d ldp ndi, chi cd 35% tning dupe phat hien d ldp can[12]. Kdt qud trong nghien ciiu nay cho thdy EFC phdt bidn tning giun san va don bao tdt ban EF cua Suzuki, 1981. Ky thuat EFC trong nghien ciiu nay da thay bdng lpc qua 2 ldp gac, khdng nia va chi ly tdm 2 ldn bang lpc qua 3 ldp gac, nia lai 3 ldn va them mdt budc ly tam lpc nia lai lan thii ba. Nhu vay lupng chdt ba va can thiic an trong phan gidm di, lupng tning giun sdn mdt di do ndi tren ldp can ba sau khi ly tam ciing giam. Didu chinh ndng dp pH cua dung dieh formalin 10% nhdm tao mdi trudng tdi uu eho ky thudt. Lpc nia lai lan thii 3 nhdm lam tning sach va rd ban phuang phdp EF ddi chiing, vi trudng sang hon dd phdt hien tning.
Ky thudt EFC phdt hien triing giun sdn cao ban ky thuat EF thudng va Kato Katz. Ty
le nhidm triing giun sdn theo 3 ky thudt tuong iing: tning giun dua la 8,4%, 5,3% va 5,3%,
triing giun tde 29,8%, 23,7% va 19,8%; triing giun mdc 19,8%, 16,0% vd 18,3%; tning
san Id nhd 21,4%, 14,5% va 4,6%; don bao 1,5%, 0% va 0% (sd lieu bdng 1). Ty Id nhidm
triing giun tde va sdn Id nhd bdng 3 phuang phap khac biet cd y nghia thdng kd (dp tin cay
95%, p < 0,05)
Vdi dung dich dem formalin 10% dupe dieu chinh bang hai dp pH = 3 va pH = 4 cho kdt qua khac nhau. Trong 101 mau dupc phan tich so sanh bang EFC vdi dung dich formalin 10% pH 3 va pH 4 cho thdy ty le nhiem trirng giun sdn phdt hien vdi PH 3 cao hon mdi trudng pH 4. Ty Id nhiem tning giun sdn theo hai ky thuat tuong iing: tning giun diia la 7,9%
va 5,0%, giun tdc la 24,8% va 19,8%, giun mdc la 25,7% va 22,8%, san la nhd la 27,7% va 15,8%, va bao nang don bao Endolimax nana la 2,0% va 0%. Sir khac bidt nay chi cd y nghia thdng kd vdi trirng giun tdc va giun moc, dp tin cdy 95%, p < 0,05.
Phuang phap xet nghiem phan true tiep chi phat hien dupc khi trong 1 gam phan cd trdn 500 tning, cdn trudng hop dudi 300 triing thudng kho phat hien. Vi vay cac phuang phap xet nghiem phong phii tning trong phan chinh xdc ban va dupc iing dung phd bien, ddc bidt phuang phap lang can Ether-Formalin. Phuang phap Kato-Katz (WHO, 1991) dd dp dung tai thuc dia vdi sd lupng mdu ldn, ddng thdi de dang phat hien tning giun sdn. Day la mdt phuong phap tien lpi khdng ddi hdi hod chdt va vat dung tdn kem, nhanh va de thyc hien.
Tuy nhien, lai khdng phdt hien bao nang dan bao va du triing. Ciing cd the dp dung de dang tai thuc dia va Labo nhung cdc phuang phdp ndi lai phai kiem tra lai ty trpng ciia dung dich [11]. Mat khde tning bi bien dang trong mdi trudng nudc mudi, khd phat hien dupe trirng san Id ldn va eac loai trirng cd ty trpng ldn. Trong khi dd phuang phdp ldng can EF khde phuc dupe nhupe didm nay bdo ddm dupc hinh dang cua tning. cd the bdo qudn dupc lau ban va tap trung dupc triing tdt hon. Vd ca ban ky thudt EF dua tren nguydn ly triing ky sinh triing va bao nang dan bao ndng hon ty trpng ciia dung dich formalin 10%. Mac dii ether la mdt trong sd cdc chdt hda tan md dupc su dung phd bidn han trong viec tap trung phdt hien tning giun san [3]. Tuy nhien, khi ether bi phai ra dnh sang nd rdt de chdy va bdc hai cao, thanh chdt gay me va dang perosides gay nd. Do dd can sii dung cdn than trong Labo ky sinh triing tranh xa liia, chdt oxy hda, idt va cio [4].
VI nhirng nhupe diem nay nen da cd mdt sd phuang phdp cai tien thay the diethyl ether bang chdt tdy nia, Tween 20^*^ hay ethyl acetate. Tuy vdy, cdc phuang phdp nay deu cd nhirng han chd va nhidu can ban phuong phdp EF [1]. Vdi EFC chung tdi thdy lupng can it hon va trong ban vi vdy khd ndng phdt hien triing giun sdn va bao nang don bao tdt ban so vdi hai phuang phdp ddi chiing (bang 1,2).
5. KET LUAN
Day la mdt phuang phdp cd thd dp dung trong nhiing nghien ciiu vd hieu qua didu tri va kidm tra khdng thudc giun sdn, ciing nhu trong nghien ciru mdt sd benh giun sdn ma vide thu hoi tning giun sdn trong phan vdn dang cdn la van de. Hon niia, vide iing dung ky thudt nay hiia ben dp dung tai cac vung da cd chuong trinh phdng chdng giun sdn nhieu ndm, noi ma ty le nhidm va cudng dp nhiem giun sdn da gidm.
TAI LIEU THAM
K H A O1. Ahmadi NA, Pakdad K (2007). Tween as a Substitute for Diethyl Ether in the Formalin-Ether Sedimentation technique. Iranian J Publ Health, Vol 36, No. 4:91-95.
2. Bo Y te, 2006. Huong dan chan doan va dieu trj mot so benh giun san a Viet Nam.
3. Cheesbrough M (1998). District laboratory practice in tropical countries, part 1. Cambridge University Press, Cambridge.
4. Erdman DD (1981). Clinical comparison of ethyl acetate and diethyl ether in the formalin ether sedimentation technique. J din. Microbiol, 14:483-85.
5. Flores A, Esteban JG, Angles R, Mas-Coma S (2001). Soil-transmitted helminth infections at very high altitude in Bolovia. Trans R Soc Trop Med Hyg, 95:272-77.
6. Kightlingger L, Kightlinger MB (1990). Examination of faecal specimens by the formahn-detergent technique. Trans R soc Trop Med Hyg, 84:417-18.
7. Lewis RJ Sr (1997). Hawley's Condensed chemical dictionary. \3*ed. Van NostrandReinhold, New York.
8. Montresor A, Crompton D. W.T, Bundy D.A.P, Hall A and SavioH L (1998). Guidelines for the evaluation of soil-t transmitted helminthiasis and schistosomiasis at community level. WHO Geneva.
9. Noriji Suzuki. Color atlas of human helminth eggs. JAPC & JOICFP. Tokyo Japan, 1981.
10. Ritchie L.S (1948). An ether sedimentation technique for routine stool examinations. Bull U.S. Army Med Dept, S:326.
11. Tai lieu tap huan, Dac diem djch te, benh hpc, dieu trj va ky thuat chan doan trong phong chong mot so benh giun san chfnh o Viet Nam. Dir an phong chong giun san nam 1998 - Bp Y te.
12. Tanaka K, Uga S (2008). Evaluation of the formalin-ether sedimentation technique for improving the recpvery efficiency of parasite eggs. Oral presentation No. 12. International conference of parasitology in Kore 2008, page 35
13. WHO, 1994. Bp tranh chan doan ky sinh trung duong rupt.
EVALUATION ON THE DETECTIVE EFFICIENCY OF PARASITE EGGS BY THE ETHER-FORMALIN SEDIMENTATION MODIFIED TECHNIQUE
Nguyen Thu Huong, Nguyen Thi Viet Hoa, Le Xuan Hung National Institute of Malariology, Parasitology and Entomology
Abstract:
The ether-formalin sedimentation technique was first introduced by Richie in 1948.
Now, it is a well-known fecal examination technique in the parasitology study. The ether- formahn technique can detect both helminth eggs and protozoan cysts simultaneously. The ether-formalin modification technique has improved the recovery efficience and amounts of parasite eggs. The results clearly show that the detective efficience of ether-formalin modifies is better than the original ether-formalin technique. This modify technique was replaced number layer of the gauze (one layers by three layers), filtration and washing (three times), and re-filtration of the plug. As results, use of three layers of gauze instead of one layer was effective for decreasing the sedimention and the reduced amounts of contaminants made microscopic examination easier. This technique can be applied to study on efficiency of mass- treatment and resistance to anthelminthie. Further, investigation into improvement of egg recovery is unstill required in where as known as reduced of prevalence and intensity.
Can bp phan bien PGS.TS Nguyen Van De Truang dai hpc Y ha Npi
TRAO DOI - HOC TAP
PHAN TICH BANG THONG KE CAC SO UEU DICH TE (tiip) Su- dung Epi-info phan tfch ket qua
Trinh Dinh Tuong
- Lenh tfnh tan so FREQ
Lenh nay diing de tinh tan so xuat hien cac gia tri trong mot biln va ti le (%) eiia no sovai tong so cac gia tri da co trong bien
Nguyen ly: Freq <ten bien>.
Lenh nay cho ket qua la mot bang so lieu co 4 cot Cot 1 ten bien thong bao ve cac gia tri cua bien Cot 2 la tan so xuat hien cua cac gia tri
Cot 3 la ti le % cua cac gia tri so vai tong so ban ghi.
Cot 4 la ti le % tieh luy.
Vi du: Ta dang lam viec vai file so lieu oswego.rec a tren.
Muon tinh tan so xuat hien theo giod tinh (sex) thuc hien nhu sau:
Tir cua so lenh go dong lenh sau: freq sex an phim enter chuong trinh se dua ra ket qua tai eua so man hinh sau:
Epi6 Er^iish Version
Dataset:
Criteria:
SEX
C:\ePI6\0SWEG0.REC i l All records selected
=:> read '
=> freq SEX Freq Percent F 4«i M 3d \ ^B.7y.
41.3/^
Cum.
im.0y.
recoras.
Output - Screen
rree memory:
Time: 0.0i
Total 75 100.0X
EPI6> read EPI6> freq SEX
F l - H e l p F2-Commands F3-Variables F4-Rrnuiqe F 5 - P r i n t e r on F9-DQS F10-(
SEX I Freq Percent Cum.
+
F I 44 58.7% 58.7%
M I 31 41.3% 100.0%
+
Total I 75 100.0%
- Lenh tao cac bang tinh TABLES
Lenh tables dugc diing de tao bang tinh tan so xuat hien cac gia tri ciia mpt bi6n so voi cac gia tri ciia bien thu 2.
So sanh sir khac nhau cac m§u quan sat
Nguyen ly: tables <ten bien 1> <ten bien 2> <ten biin 3> (phdn bd)
Lenh nay eho ket qua mot bang
Hang dong thong bao ten bien va cac gia tri cua no theo thii tu dong Hang cot thong bao ten bien 2 va moi cot la mot gia tri ciia no.
Cac 6 la tan so cua cac gia tri bien 1 vai gia tri ciia bien 2.
Vi dii vai tep so lieu tren.
Til cua so lenh ta go dong lenh: tables sex ill r6i an enter, chuang trinh se tinh ket qua nhu sau:
SEX
ILL I F M l Total + 1 30 16 I 46 - I 14 15 I 29 Total I 44 3 1 1 75
.•:'-J ' Single Table Analysis
Odds ratio 2.01 C o r n f i e l d 95% c o n f i d e n c e l i m i t s f o r OR 0 . 7 0 < OR < 5 . 8 5
Maximum l i k e l i h o o d e s t i m a t e o f OR (MLE) 1 . 9 9 E x a c t 95% c o n f i d e n c e l i m i t s f o r MLE 0 . 7 0 < OR < 5 . 7 6 E x a c t 95% M i d - P l i m i t s f o r MLE 0 . 7 7 < OR < 5 . 2 5 P r o b a b i l i t y o f MLE >= 1 . 9 9 i f p o p u l a t i o n OR = 1.0
0 . 1 1 3 2 4 3 1 9
RISK R A T I O ( R R ) ( O u t c o m e : S E X = F ; E x p o s u r e : I L L = + ) 1 . 3 5
95% confidence limits for RR 0.88 < RR < 2.08 Ignore risk ratio if case control study
Chi-Squares P-values
Uncorrected: 2.11 0.14679237 Mantel-Haenszel: 2.08 0.14951418 Yates corrected: 1.46 0.22620244
