- Giai doan thur ba tir 35 ngay 63 hoac 70 ngay sau khi nhiem 6 giai doan nay, ca'u true va churc nang ciia cac tuyen bi mit di din dan.
4.CHAN DOAN BENH TIEN MAO TRUNG 1 Ch&i doan lam sang
4.1. Ch&i doan lam sang
Cac bieu hien lam sang dac trung cua benh tien mao trung d trau, bo, ngua khong phai luc nao cung phat hien duoc. Rat nhieu gia sue mang benh nhung kho phat hien cac trieu chiing dac trung, nhat la ddi vdi nhung gia sue mac benh tien mao trung man tfnh.
Ddi vdi gia sue mac benh d the ca'p tfnh, cac bieu hien benh dac trung la sdt cao, bo an, co trieu chung than kinh (dien loan) va chet nhanh. Trau bi benh man ti'nh co the thay trieu chiing: sdt gian doan, gay com, thieu mau keo dai, viem giac mac, phu thung d bung va chan sau, chet do kiet siic (Pham Sy Lang, 1982). Trieu chiing say thau cd the thay d trau, bd bi benh tien mao trung (Nguydn Dang Khai, 1995; Kashiwazaki, 1998).
4.2. Chan doan thi nghiem
Co nhieu phuong phap chan doan tien mao trung trong phong thi nghiem, muc dich la phat hien tien mao trung trong mau gia sue. Tuy tung trudng hop benh, tuy dieu kien ma co the’ lam cung luc mot sd phuong phap hoac lua chon mot phuong phap phu hop va co do chinh xac cao.
4.2.1. P huang phap phat hien tien mao trung true tiep
Muon phat hien tien mao trung true tiep, co the: ap dung nhung phuong phap sau: * Phuong phap xem tuoi (Direct smear)
Khi sdt, trong mao quan ngoai vi cua gia sue mdc benh tien mao trung thudng co T.
evansi. Vi vay, trudng hop nay nen lay mau vung ngoai vi de xem tuoi.
Cho 1 giot mau nho len phien kinh, day lamen len giot mau de giot mau dan theo lamen thanh mot ldp mong. Soi dudi kmh hien vi (do phong dai 10 x 20) de’ phat hien tien mao trung sdng. Neu co, thay hong cau dat tu ben no sang ben kia, chiing to T.
evansi chuyen dong trong huyet tuong, lam xao dong cac hong cau.
* Phuang phap nhuom Giemsa tieu ban mau kho (Romanovsky) - Phuang phap giot day
Dat 1 giot mau to vao giua phien ki'nh, dung mot goc cua dau mot phien ki'nh khac ria tron giot mau vdi dudng ki'nh khoang 1 -1 .2 5 cm. De kho tu nhien trong khoang 1 gid. roi cd dinh bang con Methanol, nhuom Giemsa [ 1 giot Giemsa + 1 ml duns dich
PBS (Phosphat Buffered Saline) pH = 7,2 ] trong 25 phut. Rua tieu ban dudi vdi nude chay nhe, de kho r6i soi dudi kmh hien vi (dd phong dai 10 x 100 hoac 10 x 90).
Hinh 122. T. evansi (tieu bdn mau nhudm Giemsa)
Phuong phap nay co nhugc diem la de lam ton thuong tien mao trung, do do kho phan biet dugc cac loai khac nhau trong trugng hgp nhiem nhieu loai tien mao triing cung mot luc.
- Phuong phap giot mong
Dat 1 giot mau cach 1 dau phien kmh 2 cm, dung lamen ria mau thanh mot lcfp mong. De kho, co' dinh bang con Methanol trong 2 phut. Nhuom Giemsa trong 25 phut (1 gigt Giemsa + 1 ml dung dich PBS pH = 7,2). Rira tieu ban dudi voi nuoc chay nhe, de kho, soi dudi kinh hien vi (do phong dai 10 x 100 hoac 10 x 90).
Uu diem cua phuong phap giot mong la co the phan biet dugc hinh thai cac loai tien mao trung khac nhau.
- Phuong phap lam tan hong cau
Dung dich SDS (Sodium Dodecyl Sulfat) la chat lam tan hong cau. Nho dung dich SDS, tien mao trung de dang dugc phat hien trong mau. Dung dich SDS ra't ddc nen tranh tiep xuc vdi da va tranh hut bang pipet. Dung dich SDS de bao quan 6 nhiet dd thudng trong nhieu thang (Van Meirvenne, 1989).
4.2.2. Phitffng phap tap trung tien mao triing
Ngugi ta da sir dung phuong phap ly tam tap trung tien mao trung bang dng Haematocrit hoac tach tien mao trung bang gen DEAE - cellulose.
* Phuong phap ly tam tap trung bang dng Haematocrit (WOO, 1971)
Cho mau dong vat nghi mac benh vao ong Haematocrit, mot dau dng dugc bit kin bang chat deo matit, mot dau dng de ho. Ly tam vci toe do 14.000 vong/phut trong 5 phut. Sau do kiem tra su tap trung cua tien mao trung tai vi tri tiep giap giua huyet tuong va hong cau (do phong dai 10 x 10).
Phuong phap nay don gian, phat hien tien mao trung tot hon phuong phap xem tuoi va nhuom Giemsa. Song, ty le phat hien chua cao.
Vai Ong Haematocrit tren, co the thuc hien phuang phap Darkground: cat ong a vi tri tien mao trung tap trung (ranh gidi giua huyet tuong va hong cau), dung pipet lay mot ft huyS't tuong o vj trf do dat len phien kmh, day lamen va kiem tra duoi kmh hien vi (do phong dai 10 x 40).
* Phuang phap tach tien mao trung bang gen DEAE - cellulose (Diethyl - Anomino - Ethyl - cellulose).
Cac thanh phan cua huyin dich gen DEAE - cellulose - PSG (PSG: Phosphat Saline Glucose) mang dien tich duong da lien ket va giu lai cac thanh phan mang dien tich am co trong mau la hong cau va tieu cau. Con lai, tien mao trung duoc tach ra cung voi dung dich PSG va huyet tuong. Bang phuong phap nay, co the tach duac tren 96% tien mao triing (Lumsden va cs, 1979).
4.2.3. Phuang phap tiem truyen ddng vat thi nghiem
Day la phuang phap pho bien, hieu qua, chfnh xac va thuong duoc ung dung nhieu de’ chan doan benh tien mao triing a Viet Nam. Phuang phap nay co uu diem la chfnh xac, do true tiep phat hien thay tien mao triing sau khi nhan chung len trong dong vat thf nghiem man cam. Song, nhugc diem cua phuong phap tiem truyen dong vat thf nghiem la khi can chan doan nhanh, voi so' lugng nhieu va thai gian ngan thi phuang phap nay khong the dap (adsbygoogle = window.adsbygoogle || []).push({});
ung dugc (Le Nggc My, 1994; Doan Van Phuc, 1994).
4.2.4. Phuang phap chdn doan huyet thanh hoc
Bang phuang phap huyet thanh hoc, co the phat hien khang the hoac khang nguyen tien mao triing. Day la cac phuang phap huyet thanh hoc dac hieu.
4.2.4.1. C d c p hucm g p h a p p h a t hien kh d n g the khdng tien m a o triing
Khi tien mao triing ky sinh, c a the vat chu sinh ra khang the dac hieu chong lai tien mao triing. Nhirng phuang phap sau cho phep phat hien khang the khang tien mao triing trong mau vat chu:
* Phuang phap ngung ket tren phien ki'nh (SAT: Slice Agglutination Test)
Hoa tan 1 giot huyet thanh gia sue nghi mdc benh vao 1 giot nuac muoi sinh ly tren phien kmh, sau do cho 1 giot mau chuot bach co nhieu tien mao trung vao, tron deu, day lamen va soi duoi ki'nh hien vi (do phong dai 10 x 20 hoac 10 x 40). Neu thay ngung ket hinh hoa cuc la (+) va ngugc lai la (-).
Phucmg phap nay dcrn gian, dd lam va co thd ap dung trdn didn rOng. * Phuong phap LATEX (Latex Agglutination Test)
LATEX la phuong phap ngung ket gian tiep, duoc dung dd phat hidn khang thd luu dong co trong mau cua gia sue mdc benh tien mao trung.
Nguyen ly cua phuong phap LATEX: khi khang nguydn bd mat T. evansi gin ldn cac hat latex ket hop voi khang the dac hieu khang tidn mao trung o trdn ban nhua, thi se xay ra phan umg ngung kd't giua khang nguyen vdi khang thd. Hidn tuong ngung kdt c6 thd quan sat duoc bang mat thucmg, va duoc giai thfch la do khang nguydn bd mat tidn mao trung duoc gan ldn cac hat latex la loai khang nguydn hum hinh, co nhidu didm quyd't dinh tfnh khang nguydn be mat (epitop surface). Con khang thd dac hidu khang tien mao trung lai co nhidu diem thu thd (receptor) tucmg umg, dac hidu vci c£c didm quyd't dinh cua khang nguyen. Do do, khi khang nguydn tidn mao trung gap khang thd dac hieu khang tien mao trung, se co hien tuong mot phan tu khang thd dac hieu lidn kd't vdi nhidu phan tu khang nguyen va nguoc lai. Ket qua la cac hat latex cung vdi khang nguyen chum lai, tao thanh dam ngung ket co the quan sat bang mat thudng. Khang nguyen dung cho phan ting la khang nguydn be mat duoc tinh che' tu chung T. evansi
khdng nhudm mau.
Phucmg phap nay cd thd ting dung de chan doan nhanh, sd luong nhidu, ty Id phat hien cao. Tuy nhidn, nhugc didm cua phuong phap nay la co the cd phan ting gia khi pha loang huyd't thanh d ndng dd tha'p 1/2, 1/4 va duong tfnh ca khi con vat khoi bdnh nhung van cdn khang thd tdn tai trong huyd't thanh. Mat khac, phai nhap khang nguyen cua nude ngoai nen gia thanh ra't cao.
* Phuong phap CATT (Card Agglutination Test for Trypanosomiasis)
Day la phuong phap ngung ket true tiep giua khang nguyen va khang the trdn ban nhua, dugc dung de phat hien khang thd luu dong trong mau dong vat nhidm benh. Nguyen ly, cac budc tien hanh phan ting hoan toan giong phuong phap LATEX. Song co diem khac la khang nguyen dung cho phuong phap CATT la khang nguydn che toan than tien mao trung, dugc tach ra tir mau dong vat benh va nhuom mau bang comassie blu, sau do ddng khd (Bajyana Songa, 1988).
Theo Luong To Thu va cs (1996), phuong phap CATT dd sir dung, cd do nhay cao gan gap ddi phuong phap tiem truyen chuot bach (73,75% so vdi 40%).
* Phuong phap khang the huynh quang gian tiep IFAT (Indirect Fluorescent Antibody Test)
Phan ting nay la phan ting huye't thanh hoc dac hieu cd do nhay cao, dugc ting dung rong rai trong phong thf nghiem va thuc dia.
Ngoai viec dugc dung lam phan ting chuan dd so sanh vdi cac phuong phap huyet thanh khac, phuang phap IFAT con dugc dung trong nghien ctiu ca'u true cua khang nguyen tien mao trung (Luckins, 1988; Davison, 1999).
Trong phuong phap IFAT, huyet thanh duong chuan duoc lay tu trau bo mac benh tien mao trung, huyet thanh am chudn duoc lay tu trau bo khoe manh, huyet thanh can chan doan la huyet thanh la'y tir gia sue nghi mac benh.
Ung dung phuong phap nay o Viet Nam, Luong To Thu va cs (1994) da che tao conjugate huynh quang tren tho khang IgG ciia bo de chan doan benh tien mao triing. D6 pha loang cua conjugate tu che sir dung cho phan ung la 1/8 va huyet thanh chuan pha loang o 1/40. Theo Luong To Thu va Le Ngoc My (1996), do nhay ciia phuong phap IFAT la 71,25%, trong khi phuong phap tiem truyen chuot bach la 40%.
De tinh che khang nguyen T. evansi dung trong phan ung midn dich huynh quang gian tiep de chdn doan benh tien mao triing a trau bo, Vuong Thi Lan Phuong (2004) da nghien ciru khang nguyen be mat T. evansi phan lap tir trau, bo o 6 tinh phia Bac Viet Nam va cho biet: da thu duoc 6 mau tien mao triing tir 6 tinh, tien hanh phan dong, phan VAT (Variable Antigenic Type) va thu duoc 26 VAT thuoc 6 kho khang nguyen khac nhau. Bang phan ung dung giai midn djch va phuong phap tham midn dich, tac gia da xac dinh duoc su thay doi khang nguyen be mat ciia T. evansi: da so cac VAT ciia cac kho khang nguyen khac nhau la khac nhau, chi co 8 VAT/26 VAT co hieu gia khang thd don gia dac hieu, co phan ung cheo voi cac VAT cua cac kho khang nguydn khac. Cac VAT troi xuat hien som trong 4 tuan Id dau nhidm benh, co tfnh khang nguyen manh co the nghien ciru ung dung che khang nguyen chan doan. Tir do, tac gia da tinh che khang nguyen theo phuong phap tach tidn mao triing de dimg trong phan ung midn dich huynh quang gian tiep, chan doan benh tidn mao triing cho do nhay va do dac hieu cao.
* Phuong phap ELISA (Enzym Linked Immunosorbent Assay)
Phuong phap ELISA la mot trong nhirng phuang phap hien dai nhat duoc ung dung de chan doan benh tien mao triing.
Nguyen ly: dung khang the hoac khang the khang Globulin (khang khang thd) co mang mot enzym (phosphatase hoac Peroxydase) duoc gan tren manh Fc, cho ket hap true tiep hoac gian tiep voi khang nguyen. Sau do, cho ca chat sinh mau vao, ca chat se ket hap vai enzym va bi enzym phan huy tao nen mau. So sanh voi mau ciia quang pho ke se dinh luong dugc muc do ciia phan ung.
Phuang phap ELISA hien dang dugc sir dung rong rai a cac nuoc tren the giai. 6 Viet Nam, Le Ngoc My va cs (1994) da budc dau che khang nguyen tien mao triing va ung dung de chan doan benh tien mao triing a nuac ta.
4.2.4.2. Cdc phuan g phap plidt hien khdng nguyen tien mao trimg
* Phuang phap ELISA khang nguyen (Ag - ELISA)
Day la phuang phap sir dung phan ung ELISA khang nguyen de phat hien khang nguyen luu dong trong mau ciia gia sue nhidm benh. Phan ung dua tren khang the dan dong dac hieu voi tien mao triing. Le Ngoc My va cs (1994) da buoc dau ung dung phuang phap nay de chan doan benh tien mao triing a Viet Nam. Tuy nhien. theo co quan nang luong nguyen tir the giai (IAEA) (1997), phuang phap ELISA khang nguyen
co do nh$y kem hom so vdi cac phucmg phap phat hien tien mao trung c6 difin. Vi vay, phuong phap nay da khong duoc pho bien trong thcri gian hien tai va tuong lai (IAEA da khong san xua't dia Ag - ELISA cho nhieu nude tren the gidi).
* Phan umg Suratex (adsbygoogle = window.adsbygoogle || []).push({});
Phan umg Suratex la phan umg ngung ket giua cac hat latex duoc g in khang thd don dong vdi khang nguyen luu dong trong mau ddng vat nhiem tien mao trung (Nantulya,
1994). Phan umg nay hien dang duoc thu nghiem va danh gia d thuc dia.
4.2.5. Phuang phap phat hien A D N cua tien mao trung bdng phan Ung PCR
(Polymerase Chain Reaction)
PCR la phuong phap hien dai nha't, mdi duoc dua vao umg dung d i chdn doan benh tien mao trung trong nhumg nam g£n ddy.
Nguyen ly: dua vao phan umg chudi Polymerase de xac dinh su co mat gen ADN cua tien mao trung trong mau dong vat nhidm benh.
Mullis va cs (1986) da thiet lap qui trinh phan ung de ung dung chan doan benh tien mao trung. Sau do, Desquesnes (1996, 2002), Masake (1997) da umg dung phan umg PCR de chan doan benh T. evansi d bo; Almeida (1998) da umg dung chan doan benh tien mao trung d de.
Phuong phap PCR co do nhay va do chinh xac rat cao. Tuy nhien, phuong phap nay doi hoi phai cd trinh do ky thuat cao, trang thiet bi hien dai nen hien nay mdi dang dupe thu nghiem tai mot so phong thf nghiem hien dai.