DIFFERENTIATION OF MA-LAYIAND BRUGIA PAHANGIBY PCR-RFLP OF ITSl AND ITS2

S Nuchprayoon', S Sangprakarnl, AJunpeel, S Nithiuthai2. S Chungpivat2 and Y Poovorawan-'

(Departmentof Parasirology. 3Department of Pediatrics. Chula Medical Research Center. Faculty of Medicine. and 2parasitologyUnit, Department of Pathology. Faculty of Veterinary Science. Chulalongkorn.University, Bangkok. Thailand

Abstract. Lymphatic has been targclCdby the World Health Organization for elimination by the year 2020. Malayan filariasis. caused by Brl/gia lIIa/ayi,is endemic in southern Thailand where domestic cats serve as a major reservoir host. However, in nature, domestic cats also carry B. pahal1gi infection. In addition to chel11othempyand vector control. control in reservoir hosts is necessary to achieve the elimination of the disease. Thereforc. differentiation between B. ma/ayi and B. pahal1gi in the cat reservoir will help the lymphatic control program (0monitor and evaluate the real disease situation. It is difficult to differentiate these t\VOBrugia species by microscopic examination. The technique is also time-consuming and reyuires expertise. We employed the' polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) technique of internal transcribed spacer regions, ITS I and 11'52.ofribosol11alDNA (rDNA) to differentiate B. lIIa/ayifrom B.pahallgi species. Amongthe restrictionenzymes tested.only the PCR product onTS I digested with Ase I could differentiate B. ma/ayi from B. pa!Jallgi. This PCR-RFLP technique will be useful for control programs for monitoring and evaluating reservoirs.

INTRODUCTION species, such as B. pahangi -(Nithiuthai and Chungpivat, 1992; Chungpivat and Sucharit, 1993). Lymphaticfilariasis, mainly caused by the filarial Co-infections of both Brugia species in domestic cats Wuchereria bal1crofti and Brugia malayi, is not uncommon in Thailand. This raises the isa debilitatingand tlisfiguring disease. It is estimated possibility of misdiagnosis due to the difficulty in that I. ] billion people, 20% of the world's population, differentiating both species by the convent:,1nal in mOffthan 80 countries, are at risk of acquiring the microscopic method. Misdiagnosis would jeopardize infection. \..bile over' 120 million have already been the lymphatic filariasis control program. infected(WEG, 2000). Although Bancroftian filariasis In endemic areas. the routine method for hasbeencontrolled to a Iow level, 0.99 cases/IOO,OOO identifying microfilaria species is microscopic populatioD (Filariasis Division. 1999), Malayan filariasis is still endemic in southern Thailand. examination. based on the delineation of particular morphological features using Giemsa stain, and Lymphaticfilariasis. caused by B. malayi, nocturnal geographic location. that is, where the specimens have subperiodic type, is prevalent mainly in Narathiwat come from. Nevertheless, using this technique, it is Province. This is due to the many suitable mosquito difficult to discriminate clearly between closely related breeding sites, large swamp areas. and the existence species such as B. l11alayi and B. pahal1gi. of animal reservoir-hosts. In Thailand, sp Histochemical staining, to detect acid phosphatase arethe main mosquito vectors of B. malayi (Filariasis activity, could overcome the problem (Yen and Mak. Division,1998). Domestic cats are important reservoir 1978: Chungpivat er ai, 1990: Nithiuthai and hosts for B. malayi (Palmieri et al. 1985; Phantana et Chungpivat, 1992: Chungpivat and Sucharit. 1993). ai, 1987:Kanjanopas et ai, 2001; Chansiri et ai, 2002). However. this technique needs fresh samples to yield Therefore.besides chemotherapy and vector control, the best results. Furthermore, both staining methods theSuccessfulelimination of lymphatic filariasisshould require expertise to identify and confirm the species. include the control of reservoir hosts. Not only B. llzalayicaninfect domestic cats, but also anotherfilaria! Molecular analysis has been introduced as a new tool to distinguish parasite species. Ribosomal DNA (rDNA) has been a valuable tool to discriminate closely Correspondence:Dr Surang Nuchprayoon, Department related species among several eukaryotic organisms, of Parasitology. Faculty of Medicine, Chulalongkorn including nematodes. The application of internal University,Bangkok 10330, Thailand. transcribed spacers (ITS) to identify the organism has Tel:66 (0) 2256-4387; Fax: 66 (0) 2252-4963 received the most attention by nematologists during E-mail:[email protected] the past decade (Gasser et al. 1994; 1996; Zhu et al,

Vol34 (SuppI2) 2003 67 SOlITHEAST ASIAN .J TROt' MED PUBLIC HEALTH

1998;Almeyda-A11igaset aI,2000; Conole et aI,200 I). Primers design Studies of peR-linked restriction fragment length The forward primer for the ITS 1 (FU-F) \Vas polymorphism (PCR-RFLP) profiles of the nematodes' clesigned from the conserved sequence of filarial ITS regions have provided data on diversity, parasites reported in the Genbank database (Fig 1). The as well as ,the critical taxonomic character useful for reverse primer for the ITS 1 (Di5.8S 660-R) and the species colnparison and identification (Gasser et al. forward primer for the ITS2 (Di5.8S 558-F) were 1994. 1996). We report here the results of PCR-RFLP designed from the rep0l1ed 5.8S rDNA sequence of D. of ITS I. and ITS2 to differentiate B. lIIalayifrom B. illlmitis(AF217800). The reverse Plimerfor ITS2 (FU- pahmzgi. R) was designed from the 28S rDNA sequence of D. i11l11litis(AF217800). All oligonucleotide primers were MATERIALS AND METHODS purchasedfromthe Bioservice Unit. NSTDA. Bangkok

Polymerase chain reactions Specimen collection In order to obtain sufficient DNA for RFLP study. In co-operation with the Filariasis Division, semi-nested PCR for-ITS 1and ITS2 were performed. Department of Communicable Diseases Control. Ministry of Public Health, Thailand. collection of First PCR ITSl-5.8S-ITS2 region. The entire human blood specimens was performed as previously ITS 1-5.8S-ITS2 region (Fig 1)was amplified by PCR described (Triteeraprapab and Songtrus, 1999; using FLI-F and FL2-R oligonucleotide primers. The Triteeraprapab et aI, 2000; 200 I; Nuchprayoon et aI, hot-start PCR reaction was performed in a 25 III 200 I). Two milliliters of venous blood from domestic reaction containing PCR buffer (10 mM Tris-HCI, pH cats infected with B. Illalayi or B. pahangi were 9.0, 1.5 mM MgC12. 50 mM KCI) (Amersham obtained from experimental cats at the Parasitology Pharmacia, Freiburg, Gelll1any);200 ~tMeach of dATP. Unit, Department of Pathology. Faculty of Veterinary dCTP. dGTP and dTTP (Proll1ega,Wisconsin, USA): Science, Chulalongkorn University. Dirofilaria 0.625 units Taq DNA polymerase (Amersham ill/mitismicrofilariae were obtained from infected stray Pharmacia); 5 pmol of each primer (FLl-F and FU- dogs' blood. Blood from healthy volunteers, non- Ri; and I IIIDNA template. After incubation at 94cC infected domestic cats and dogs were used as the for 5 minutes. amplification was carried out for 5 cycles negative control. This study was approved by the with the following temperature cycling parameters: Ethics Committee of the Faculty of Medicine. 94"C for 30 seconds of denaturation; 58'C for 30 Chulalongkorn University. Bangkok. Thailand. seconds of annealing; and noc for 90 seconds of extension; followed by 30 cycles of temperature All filarial parasites were identified and the species cycling parameters at 94 QC for 30 seconds of confirmed by Giemsa staining. and special staining for denaturation; 55'C for 30 seconds of annealing, and acid phosphatase activity (Yen and Mak, 1978; 72"C for 90 seconds of extension. The final Chungpivat et aI, 1990; Nithiuthai and Chungpivat, amplification cycle included an additional 10 minutes" 1992; Chungpivat and Sucharit, 1993). extension at n'c.

DNA extraction Second PCR for ITSl region. The ITS 1 region The extraction method was modified from a was amplified by using I III of PCR product froll1the previously reported protocol (William et aI, 1996). first PCR as a DNA template. The PCR reaction wa" Two hundred and fifty microliters of each blood sample performed in a 50 1-11reaction containing PCR buffer. were added to a 1.5 ml microfuge tube and mixed with as described above, with 5 pmol of each primer ofFLl- 750 III ofTE buffer (l0 mM Tris-HCL pH 8.0, 1 mM F and Di5.8S 660-R. Amplification was canied out EDTA, pH 8.0). The mixture was centrifuged and the for 5 cycles with the following temperature cycling supematant was discarded. The pellet was washed with parameters: 94'C for 30 seconds of denaturation; 58'( 750 IIIofTE buffer, pH 8.0 and resuspended in 500 III for 30 seconds of annealing; and noc for 45 seconds of red cell lysis buffer (RCLB; 1 M sucrose, 10 mM of extension. followed by 30 cycles of temperature Tris-HCI, pH 7.5. 5 mM MgCI2, ]% Triton X-lOO) cycling parameters: 94 QC for 30 seconds of twice. After centrifugation, the supernatant was denaturation. 55°C for 30 seconds of annealing; and discarded, 400 IIIofDSP buffer (20 mM Tris-HCl, pH n"c for 45 seconds of extension. The final 8.0, 50 mM KCI. 2.5 mM MgCI2, O.Wc Tween 20. amplification cycle included an additional 10 minutes 150 Ilg/ml Proteinase K) were added and incubated at extension at n'c. 65"C for 3 hours. Enzyme activity was inactivated by incubation at 90QCfor 10 minutes. Second PCR for ITS2 region. The ITS2 region

68 Vol34 (Supp12) 2003 PCR-RFLP OF ITS 1 AND ITS2

wasamplified by using 1111of PCR product from the resuspension in 10 III sterile distilled water, I 111of firstPCR as a DNA template. The PCR reaction was PCR product was digested with 5 units of each performed in a 50 111reaction containing PCR buffer restriction endonuclease, according to the with5 pmol oreach primer, Di5.8S 558-F and FL2-R. manufacturer's protocols (New England Biolabs. After incubation at 94°C for 5 minutes, amplification Massachusetts. USA). The digestion was incubated at was carried out for 35 cycles with the following 37"C for 3 hours. The following enzymes were temperature cycling parameters: 94cC for 30 seconds evaluated; Ase I. Ace I, HinfI and Rsa I. Analysis of ofdenaturation; 55°C for 30 seconds of annealing; and DNA fragments was performed by 2-2.5% submarine noc for 45 seconds of extension. The final agarose gel electrophoresis. amplificationcycle included an additional 10minutes' extension at 72"C. RESULTS

Restriction fragment length polymorphism (RFLP) Oligonucleotide primers were designed based on After precipitation of each PCR product and the reported conserved sequences of 18S,5.8S and 28S

I FLl -F I ... 5' 1 gtgaacctg cggaaggatc attatcgagc ttcaacaaac aacaaacaca tcatcatcat 61 catcattatt actacttcta ctactactac tactactact aatactaata ctaatactac 121 ta.ttaacatc attatgctgc ttattttcat tctttttcaa ataatgcaag aatgaaatag 181 aatgaatgaa tgatgtttaa tagattaata gatgaatagt tagtagtagt tagtagttag 241 ttgatagat agataaatga aatgatgatg aaatattttt attcgatcaa t.tg.a.atataa 301 acggtgatat tcgttggtgt ctatatttta tctaagttat cgcctaaccg tcgatagcga 361 421 ttttagctag tatgctacca a DiS.8S558-F cacaca<:; atacatacaa taatatgata 481 taattttattgaagataaa ttataataatatgatagctt ta" . ]tgctcaatagcgg gggatcacctttaagtagac ggctcgtggattaataagca 541 tcgatgaaga acgcagctag ctgcgataaa tagt cgaat tgcagacgca ttgagcacaa 601 agatttcgaa tgcacattgc 661 caatatcgat cagattatca accatcgggtaataatatac tgattcccgacaaaattgat tggiacgtctaca atl . ggttgagggttg 721 atatttt tggatatatt atttgttaaa attaattcat caggtg Di5.8S 66J-R ta 781 atcattgaaa aatgacai1ai1 atcccctgat taagtataat aaaaat ag 841 gcaaattttt acttacaaaa tattacatat tggaataaat aatttaatct ttcatatata 901 taactgtgat acatcataaa t.atatatata gcaaaagaaa gatataaatt attatcattt 961 tttttgatca ttattgctat aattattatc ctgcttgttt aactataata atagcaatga 1021 atgaatcaaa atttaaatga tacgttaaaa tccaaccaat atgttataaa tt.ttctttca --., 1081 tttttgacct caactcagtc gtgattacc gctgaattta o:.gcata taac t.a ,;)

I FL2-R I

FLI-F DiS.SS 55S-F DiS.SS 660-R FL2-R --+ --+ ~ ~

188 5.8S 28S }

ITS 1

667 bp ITS!

570 bp

Fig 1- Forward and reverse primers for ITS 1and ITS2. The FLI-F primer is conserved in filarial parasites. All other primers were designed from the D. immitis 5.8S and 28S rDNA sequence (AF2 I7800). FLI-F and Di5.8S 660-R are primers for the amplification of ITS 1;.Di 5.8S 558-F and FL2-R primers are for ITS2.

Vol34 (SuppI2) 2003 69 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH

rDNAof filarialworms(B. l1Ialayi, W. bal1cJ{~fiiand it requires skill and experience to differentiate among D. illllllitis). Nevertheless, not all rDNA sequences of closely-related species. Histochemical stain of acid the selected filarial worms were completely reported. phosphatase activity is another useful technique for The available filarial rDNA sequences from the online species identification. It clearly discriminates different database (Genbank) were as follows: 18S rDNA from filarial nematode species (Yen and Mak. 1978; B. lllalayi (AF036588), W. bancrofti (AF227134). and Chungpivat et af. 1990: Nithiuthai and Chungpivat, D. illlmitis (AF217800): ITS!, 5.8S and ITS2 sequence 1992:Chungpi\'at and Sucharit, 1993). However. the from D. illllllitis (AF217800); partial 28S rDNA enzymatic activity is rather sensitive to light and sequence from D. illllllitis (AF2 I7800) and B. 111alayi requires fresh specimens. As a result, PCR-RFLP of (AF499 130). The FLI-F primcr was designed from the ITS regions may be an alternative method to the conserved region at the 3-end of B. l7Ialayi. W. differentiate closely-related species. which are bancrofti and D. illll7litis18SrDNA (Fig I). The FL2- indistinguishableby morphology(Gasseretal. 1998). R primer was selected from the beginning 5' end of D. Furthermore. PCR-RFLP could be used to identify immitis 28SrDNA, whichwasalso conservedamong parasites in developmental stages (Gasser and Chilton. the reported 28S rDNA from other nematodes. After 1995: Newton et al. 1998: Almeyda-Artigas er (//, the first round of PCR, very faint DNA bands of ITS 1- 2000) in different geographic distributions (Gasser and S.8S-ITS2 were observed. Subsequently. semi-nested Chilton, 1995; Ramachandran et aI, 1997). PCR was performed to obtain a high yield of PCR The ITS I and ITS2 regions of rDNA are useful products. Two internal primers, Di5.8S 660-R and for investigatingsome variations among closely-related Di5.8S558-F.weredesignedfromtheD. illll1litis 5.8S species(Gasseret aI, 1996;Ramachandranetal. 1997). rDNA region (AF217800) in order to amplify the ITS 1 The ITS regions are flanked by conserved rDNA genes, and ITS2 regions, respectively. and the sequences can be use? 10 design primers to amplify the intervening regions by PCR. Moreover, PCR-RFLP patterns onTS! and ITS2 the presence of multiple copies provides a large number The 667 bp band of D. illll1litisITS 1was amplified of target sequences for PCR in most organisms (Long as expected(Fig 2A). However.both B. lIlalayi and and Dawid, 1980). As a result. the ITS regions of B. pahangi ITS1hadsmallerITSI sizesof about590 filarial nematodes are suitable targets for amplification bp. Incontrast,the ITS2from D. il1llllitis was570bp, and detection, even in mildly infected individuals. and for B. lllalayiandB. pahangi ITS2 about 650 bp Although the first round PCR was inadequate to (Fig2B). BothB. malayi from humansand domestic provide a detectable amount of PCR products. a cats showed the same size of~CR products and similar subsequent semi-nested PCR has been utilized to digestedpatternsforITSI andITS2(Fig2A, B). There increase the sensitivity for the detection of the ITSI were 2 bands of about 290 bp and 300 bp from the Ase and ITS2 regions. Since the filarial DNA was extracted I digestedB. pahangi ITS1(Fig2A),whileabout 140 from whole blood specimens of infected individuals. bp, 160 bp and 290 bp bands were from the B. lIlalayi host genomic DNA might possibly interfere with the digested ITS I. PCR-RFLP of ITS 1 with Acc I and PCR reaction. However. the bases at the 3- end of with Hinf I did not differentiate B. lIlafayi from B. both internal primers designed for semi-nested PCR pahangi. Similary, the PCR-RFLP of ITS2 with Rsa I of ITS 1 and ITS2 were not complementary to human and with Ase I did not discriminate either Brugia rDNA (U 13369). Therefore, no ITS 1or ITS2 products species. were amplified from the semi-nested PCR of the non- PCR-RFLPof D. il1ll11itisITSI by Ase I showed infected human blood samples (data not shown). There the 455 bp and 212 bp predicted products. All other was no reported sequence of ITS 1 and ITS2 from digested ITS I and ITS2 showed the predicted PCR- domestic cats and dogs. Nevertheless, no PCR RFLPpatterns. We demonstrated that the semi-nested products of ITS I and ITS2 were amplified from the PCR-RFLP of ITS 1 with Ase I could differentiate B. non-infected blood samples of domestic cats and dogs l11alayi, B. pahal/gi and D. il11l11itis. (data not shown).

The PCR-RFLP profiles of domestic cat B. /l/alayi DISCUSSION and human B. /l/afayi ITS I and ITS2 were similar for all restriction endonucleases used in our study. Our The conventional method used for the detection results supP011edthe suggestion that domestic cats play and species differentiation of lymphatic filarial an important role as animal reservoirs of B. malayi. as parasites is the identification of microfilariae by reported previously by studies of microfilarial Giemsa stain. Although this method is not expensive, morphology, periodicity, PCR-RFLP of Hha I repeat

70 Vol34 (SuppI2) 2003 PCR-RFLP OF ITS I-\ND ITS2

A..ITS 1

Hinf I .Ace I As'e I - --

r...1 Undigested 1....1 Digested i\'I Digested 1\'1 Digested hEm rBm Bp Di hBm cBm Bp Di hBm rBm Bp Di hBm cBm Bp Di

,455bp 'l /212 bp J Di I' " Di:6(;7bp I' // L,. /' :~ Bm, Bp: 590 bp I /- 300 bp 1.

" 290bp IfBp /" loObp " Bm 140 bp .-r

B. IT S2 Rsa I L4s'e I

l\'1 Undigested rill Digested M Digested hEm cEm Bp Di hEm rBm Bp Di hEm cEm Bp Di

Bm,Bp: 050bp L. r Di:570bp

Fig 2- PCR-RFLP of B. malayi and B. pahal1gi ITS I (A) and ITS2 IB). M: 100 bp ladder molecular weight marker; hBm: human B. malU\'i; cB m: cat B. malm'i; Bp: B. pahal1gi; Di: D. immitis; Asf' I. Ace!. liil1fl and Rsa I are restriction enzymes used for the digestion of ITS 1 and ITS 2 PCR products. genes and PCR ofTrans-spliced Leader Exon I (SLX) been shown that domestic cats in Thailand can carry (Chansiri et al. 2002). not only E, jJahangi and E. 17Il/layi,but also D. inuuitis (Chansiri et 01, 2002). Further study, for the field In summary. we demonstrated that the PCR-RFLP application of the PCR-RFLP method to monitor and of ITS 1, digested with A.I'e1. could differentiate E, evaluate infections in the reservoir hosts, would help l11alayi,B. pahangi and D. il11l11itis.Recently. it has the on-going lymphatic filariasIs control programs.

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ACKNOWLEDGEMENTS 1999 (in Thai). Gasser RB. Chilton NB, Hoste H, Stevensor. LA. This study was supported by the grants forgraduate Species identificationoftrichostrongyle nematodes students in public universities, the Graduate School by peR-linked RFLP./nt J ParasitoI1994:24:291. and Molecular Biology Funds, Research Affairs. 3. Faculty of Medicine. Chulalongkorn University. Y Poovofawan was a Senior Research Scholar of the Gasser RB, Chilton NB. Characterisation of taeniid Thailand Research Fund. We would like to thank Dr cestode species by PCR-RFLP of ITS2 ribosomal Saravudh Suvannadabba, Dr Suvith Thampaolo. Ms DNA. Acta Trop 1995;59:31-40. Kobkarn Kanjanopas, Ms Sumas Loymak, officers at Gasser RB. LeGoff L. Petit G, Bain O. Rapid the Filariasis Division, and regional officers. for their delineation of closely-related filarial parasites support during specimen collection. We are th:mkful using genetic markers in spaceI' rDNA. Ac/(( Trop to 1\1sWoraporn Sirivichayakul and all staff in the 1996:62:143.50. Parasitology Unit, Department of Pathology. Faculty of Veterinary Science and staff in the Departmcnt of Gasser RB. Manti JR, Bao-Zhen Q. Polderman AM. Parasitology, Faculty of Medicinc, Chulalongkorn Nanscn P. Chilton NB. A mutation scanning University for their technical help. We also appreciate approach far the identification of Dr Benjawan Sichnasai and staff from the Rabies species and analysis of population variation. Mo! Control Section. Bangkok Metropolitan Area for their Biochem Parasilol 1998;92:303-12. kind assistance. Kanjanopas KoChoochote W, Jitpakdi A, el (/1.Brt/gia REFERENCES nwla.vi in a naturally infected cat from Narathiwat Province. southern Thailand. Sowheasl Asian J hop A-fedPublic Health 2001;32:585-7. Almeyda-Artigas RJ, Bargues MD, Mas-Coma S. ITS- 2 rDNA sequencing of species Long EO. Oawid lB. Repeated genes in eukaryotes. (Nematoda) and elucidation of the species causing Annll Rc!' Biochel7l 1980: 49:727-64. human in the Americas. J Parasifo/2000;86:537-44. Nithiuthai S, Chungpivat S. Lymphatic filariasis Wmgia pahangi) in dogs. J Thai Vel Pract Chansiri K, Tejang;,ura T, Kwaosak P. Sarataphan N, 1992:4: 122-33. Phantana S. Sukhumsirichart W. PCR based Nc\vton LA, Chilton NB, Beveridge I, Hoste H. Nansen method for identification of zoonostic Brugia p, Gasser RB. Genetic markers for strongyliJ l11a/ayi microfilariae in domestic cats. M(J/ Cell nematodes of livestock defined by peR-based Probes 2002: 16: 129-35. restriction analysis of spaceI' rDNA. Acta Trop Chungpivat S, Nithiuthai N. Sichanasai B, 1998:69:1-15. Santiwatanatarm T. Histochemical diagnosis of Nuchprayoon S. Yentakam S, Sangprakarn S, Junpee Dirofilaria immitis microfilariae by acid A. Endemic bancroftian filariasis in Thailand: phosphatase activity. J Thai VetPracl 1990:2:195- 203. detection by Og4C3 antigen capture ELlSA and the polymerase chain reaction. J Med Assoc Thai Chungpivat S, Sucharit S. Microfilariae in cats in 2001:84:1300-7. Bangkok. Thai J VetMed 1993:23:75-87. Palmieri JR, Ratiwayanto S, Masbar S, Tirtokusumo Concle Je. Chilton NB. Jarvis T, Gasser RB. Mutation S, Rusch J, ~vlarwoto HA. Evidence of possible scanning analysis of microsatellite variability natural infections of man with Brt/gia pahangi in in the second internal transcribed spaceI' (pre- South Kalimantan (Borneo), Indonesia. Trop cursor ribosomal RNA) for three species of Geogr Med 1985;37:239-44. MelastmngY/lls (: Metastrongyloidea). Phantana S, Shutidamrong S, Chusattayanond W. Parasitology 200 I: 122:195-206. " Bmgia malayi in a cat from southern Thailand. Filariasis Division. CDC Department. Ministry of Tra/1l R SOl' Trop /v/I'd HYR 1987;81: 173-4. Public Health, Bangkok, Thailand. Annual report. Ramachandran S. Gam AA. Neva FA. Molecular 1998 (in Thai). differences between several species of Filariasis Division, CDC Department, Ministry of Slrongv/oides and comparison of selected isolates Public Health, Bangkok, Thailand. Annual report. of S. stercoralis using a polymerase chain reaction- n Vol34 (Suppl 2) 2003 PCR-RFLPOF ITS I ANOITS2

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