ABSTRAK
Mutasi titik gen pfcrt pada penderita malaria falciparum umur 1-18 tahun di Kabupaten Ogan Komering Ulu Provinsi Sumatera Selatan
Rosiana A Marbun
Temuan dalam beberapa tahun terakhir menyebutkan bahwa resistensi P. falciparum terhadap klorokuin terutama diperankan oleh mutasi pada gen P. falciparum chloroquine resistance transporter (pfcrt).Tujuan penelitian ini adalah untuk mengidentifikasi dan mendeskripsikan pola distribusi mutasi titik gen pfcrt P. falciparum berdasarkan demografi dan tofografi di Ogan Komering Ulu (OKU) Sumatera Selatan. Penelitian ini merupakan penelitian deskriptif eksploratorik pada anak berumur 1-18 tahun di 3 daerah endemis malaria yang mewakili OKU Sumatera Selatan yaitu Tanjung Lengkayap, Banding Agung, dan Belitang. Tahapannya adalah penentuan spleen rate, pengumpulan subyek, diagnosis malaria klinis, pemeriksaan mikroskopis sediaan tebal dan tipis spesimen darah, uji resistensi klorokuin in vivo, isolasi DNA, PCR, dan sequencing. Nilai spleen rate ke 3 daerah tersebut dikelompokan ke dalam kategori hipoendemis malaria. Diperoleh 171 sampel dengan malaria falciparum. Pada uji resistensi klorokuin in vitro 25 sampel gagal, dan 146 sampel berikutnya dilanjutkan uji resistensi in vivo, didapat 44 sampel yang resisten terhadap klorokuin. Berhasil dilakukan isolasi 168 sampel DNA gen pfcrt, PCR, dan sequencing, didapatkan mutasi titik pada kodon 74, 75, 76, 97, 220, dan 356 yaitu M74I, N75L, K76T, H97F, A220S, dan I356L. Pola distribusi mutasi titik di Tanjung Lengkayap, Banding Agung, dan Belitang 100% pada kodon 74,75,76, dan 356, sedangkan pada kodon 97 berturut-turut 44%, 46,9%, dan 60%, pada kodon 220 berturut-turut 40,5%, 46,9%, dan 60%. Kesimpulan adalah Distribusi mutasi titik pada kodon 97 dan 220 dijumpai paling banyak di Belitang. Didapatkan mutasi titik lokal baru pada kodon 75 dan 97 gen pfcrt P. falciparum belum pernah dijumpai di tempat lain.
Kata kunci: Malaria falciparum, klorokuin, resistensi, mutasi titik gen pfcrt
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Desertasi Mutasi titik gen pfcrt pada penderita .... Rosiana A. Marbun Sumatera Selatan. Peneelilitititanana i ini merupakan penelitia
ratorik pada anak bebeberururumumumur r r 1-1-1-181818 t tahahahununun d d di ii 3 3 3 daerah endemis m ili OKU Summatataterereraa a SeSelaSelalatatatan n n yayayaitttuuu TTaTanjnjnjununung g Leg LeLengngngkakakayayy p, Banding g. Tahapapaannnnnnyayaya aaadadadalalaahhh pepepepepepp neneneneneneennntntntnttuauauauauauu nnn n n splespspleleenenen r r ratateatee, , , pepp ngumpu sis malalaaririria a a klkllininnisisis,,, pepepepepeemememememeerirririririksksksksksksk aaaaaaaaaaannnnn m mmikmmikikikkrrorororoosksksksskss opopopisss sededediaiaiaana teb en darararahah, ah, ujujuji i i rerereeeesisisisssstststtttenenenennsisi ksisisi k k kklollololol rororookuokukukukuk iniinin inininnn v v vivvvivoivivivoooo, , , i iisososolalalasisisi DNA cinggg.. NiNiNilalalai i i spspspspsppplelelelelel enenenenn r r r ratraaatateee kkke kkkkkkkke e eee 333 3 dadadaaaerererererahahahahh t ttterterersrrsseseseseebububububuutttttt d d dikikikelelomlomompopp ka ri hhipipipoeoeoendndndemememmismmmisisss m mmmmalaalaalararaaraaiaiai .. . DiDiDiDiDippeperopepeppeppp rrrr llllehhhh h 17171717171 1 sssasasampmpelmpmpmpelelelel d d d ddenenennnngagag n n mamamalalalari
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ciiingngng, , d d didididdapapapapapatatatatakkakakakakann n n m mmutmmutututasutasasasii i i tttittitik padadadadadaa a a koodoooododododon n n n 7474, 747474,, , 75757575, 767676,, , 979797,, 2 M7774I4I, 4I,,N777555LL, LLL, , K7K7K7K7K76T66T6T6T, ,,,,H9H9H9H9H997F7F7F77F, A2A2A2A2A220202 S, dS,S, dan d ddaaaaa II35IIII353535356L6L6LL. PoPPoPoPolalalala da d ddiisiisstrtrtribusususiii m
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titikkk p p padadada a a kokokooodododododon n nn n 9797979797 d dan d ddanananan 2 222202020 d2020 d dddijijijijijuumpuuumpmpmpmpaiaiaiaiai p p p p pppaalalalaalaininngg g bababanynynyak tkan mmmutututasasasi i i titititititik kklololololololokakakakak ll lllbababababarururururu p ppadppadadada addaaaakokodokokokododododod nnn nn757575757575 d dddandddanan 9997 gegegen n n pfppcrt P pernah ddijijijumumumpapapaiii dididi t tempaemempapapapapapatt ttlalainaaann.
ABSTRACT
Point Mutations of Pfcrt Gene in Patients aged 1-18 years with Falciparum Malaria in Ogan Komering Ulu District in South Sumatra Province
Rosiana A Marbun
The findings in recent years state that the resistance of P. falciparum to chloroquine mainly caused by the mutation in the P. falciparum chloroquine resistance transporter (pfcrt) gene. The objectives of this study were to identify and describe the distribution pattern of point mutations P. falciparum pfcrt gene based on demographics and topography in Ogan Komering Ulu (OKU), South Sumatra. This study was a descriptive exploratory study in children aged 1-18 years in three malaria-endemic areas which represents OKU-South Sumatra, namely the Tanjung Lengkayap, Banding Agung, and Belitang. The stages of this research were the determination of spleen rate, collecting subjects, clinical malaria diagnosis, microscopic examination of thick and thin stocks of blood specimens, test of chloroquine resistance in vivo, DNA isolation, PCR, and sequencing. Based on the value of spleen rate, those 3 regions were grouped into hypoendemic malaria. One hundred seventy-one (171) samples were obtained with falciparum malaria observed by microscopic examination of thin blood supply. Twenty-five (25) samples failed the test of chloroquine resistance in vitro, and the next 146 samples would be proceeded to take resistance test in vivo which, later, it was found that 44 samples were resistant to chloroquine. One hundred sixty-eight (168) samples of pfcrt gene DNA were successfully analyzed by conducting isolation, PCR, and sequencing. As a result, a point mutation was found at codons 74, 75, 76, 97, 220, and 356, namely M74I, N75L, K76T, H97F, A220S, and I356L . The pattern of distribution of point mutations in Tanjung Lengkayap, Banding Agung, and Belitang was 100% in codons 74,75,76, and 356, while at codon 97 was respectively 44%, 46.9%, and 60%, , at codon 220 was respectively 40.5%, 46.9%, and 60%. This research concluded that distribution of point mutations at codons 97 and 220 were mostly found in Belitang. A new local point mutation at codons 75 and 97 of P. falciparum pfcrt gene was found. This has never been found in other places.
Keywords: falciparum malaria, chloroquine, resistance, point mutations of pfcrt gene
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Desertasi Mutasi titik gen pfcrt pada penderita .... Rosiana A. Marbun n three malaria-endemic aarereeasasas w which represents OKU-Sou
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76,,, 979797, 222222222222 0,0,0,0,0, aaaaandndndndnd 3333556, 56565 nanaaamememememelyelylyly M M M M74M74747474III,II N N NNN7575757575L,L,L,L,L,, KKKKK7676767676T,TT,T, H HH979797F,FF . The ThThe e papapatttteeeerererrn ofofofof d d dddisistrisisstrtrtribtributibibib tioioioion ion n nn ofofofofofffff ppoipppointoioii tt mmmututututatutatatatatioioioioionsnsnsnsns i i iiin in n TaTaTanjnjnjununu g g AgAgAgununung,g,g, an a anndndndndnd BBBBBBelelellitititititanananana g g g wawawas s sss 1010101000%0%0% i%% iin n n cocococodoodododoonsnsnsn 74,7774,4,4,4,4,,777575757 ,76,,7,76,6, a a andnn 3 97 wasasas r rresesespepepectctivctivivivivvvelelely elelyy y y 444444444%,%,%, 4%,%, 4 4 44666.6.6.9%9%9%, 9%9%,,,ananananand d 60d d d 60606060%,%,%,%,%, ,,, a at a a at t ttcococccc dododon n n2222220 0 0was
46.9%%%, , , anananddd 6060600%.%.%.%.%.%% TThiTTThihihihiss s s s rerereeessesesseararcharararchchchch c c con ccononononcccccclulululuuudededdededdedd d tthatatt ddddddisisistribut ons at codddononons s s 979797 a and and 2nd 2 22222022020202020 wwwwwweeeere eee mommomoostststttllylylyyyyyfffffofouoouououndndnd i iin n Bn BeBeliliitatatannng. A ne on at codonsnss 7 7 75 5 5 aaand 97 o9797 o of f P.P.P. f f falalalccipipiparararumumum p ppfcccrtrtrt g g genene e was fou
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ABSTRACT nce strains survive because ttheheeyy ycacac n nreduce accumulation of ch
Key words: malaria falciparum, chloroquine, resistance , mutation of pfcrt gene
SUMMARY
Point mutation pfcrt gene of malaria falciparum patients the group of 1-18 years old in Ogan Komering Ulu districts
South Sumatera Province
Malaria is one of the major world health problems with 300-500 million cases and 1.5-3.7 millions death annualy.There were 243 million cases and 863,,000 death and still high in 2008. According to World Health Organization (WHO) data, about 56% of world population live in endemic malaria area. In Indonesia, there are 45% of the population live in endemic malaria area also some area in South Sumatera including Ogan Komering Ulu (OKU) districts, Annual Clinical Malaria Incidence (AMI) out-side Jawa-Bali were 23.98‰ in 2006.
Annual Clinical Malaria Incidence in South Sumatera 8.7‰ and 23.48‰ in Oku
districts in 2009. Aproximately 61.4% of parasite causing malaria in Indonesia are P. falciparum also in OKU districts around 71,64% in 2008.
Chloroquine is a antimalaria agent of 4-amino quinoline group which is schizonrosidal to all kind of Plasmodium and also gametosidal to P. vivax and P. malariae. The drug were delivered to digestive vacuole to inhibit protein synthesis
ofP. falciparum.
P. falciparum resistance to antimalaria drugs are serous problems for
selecting antimalaria drugs, patient management, prolong of hospitalized and increasing of mortality. World Malaria Report (2005) showed the endemic malaria with chloroquine resistance including South and Central America, West, Central and East Africa, South Asia, Melanesia Islands and Southeast Asia including Indonesia. Data of 1996 showed that P. falciparum chloroquine resistance were found in all province of Indonesia with varieties of prevalence. The average of P. falciparum chloroquine resistance in vivo in Indonesia was 52% and 59% in vitro. P. falciparum resistance in vivo are ability of parasite to survive human host, multiplication and produce of diseases although the treatment
xxix
Desertasi Mutasi titik gen pfcrt pada penderita .... Rosiana A. Marbun
j p
and 1.5-3.7 milliooonsnsns d d deaeaeaththth a annnnnnuauaually.y.y.ThThThererere e e were 243 millio
00 death anddd s s stititillllll high h h hh ininin 2 2 2000008 A88. AccAcccororordididng tnn tto o WoWoWorlrrd Health ) data, abababououout 55656%% % ofofofofofofof w w w wworwororororldorlldldldld p p p pppopopopopopululuulululatatatattioiioioioioionnn n n lillililililiveveve in nn ennndededemimmc mal sia, theheherere are a arerere 4545454555% % %% % ofofofofof t tthetthehehhee p p popppopulopopopululululatatatition ioionnnlilililiivevvveve i i iiin n n n neneenenenendddedededeemic mimic c mamamalalalarirra ar Soooutututh h h SuSuSumamatemamamamm tetetet ra irrarara incincnnnnclulul diddidididididngngngngngggg O O OOOggannnnn K K K KKomomommeeerererinininining ggg g UlUlUlUlUlu uu uu ((((O((O(OKUKUKU) ) ) dddist l MMMalalalaraa iaiaia IIncIIIncncncncididididdenenceenenencececece ((((AMAMAMAMMMI)I)I)I)I) ouutuutut-s-s-sididde ddee ee JaJaJaJaawawawawawa-B-B-B-BBalali alalali i wwewewwww reee 23.223.39989
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Cllilinininicacacal l MaMalaMaMaMalalalalariririririaa Ina aa InInInInciciciciidededeeencncncnceeeee in Sooutouth utututhh SumaSuSuSuumammatemateteteerarara 8rara 8 8 788.7.7‰77‰‰ ‰‰‰‰ananandd d 232323 s ininn 2 2 20000009.9.9. A AprAAproxprprproxoxo imimimimatmatelatatatelelelely yy y y61.461616161.4.4.4.4% %offff p p pappaaararrar ssisisiiitetetetete c cccauausiauusisisisingngngngg m mal mmmalalaaariaiaia i i in n nI
paaarururummm a aalsolslso oo oooininininn OOOOOKUKUKUKUKU d dis dddisiii triccttts ssss araaraararououndououndndndnd 7 7 777111,64646444% % %%%iininin 2 22002200000008.8.888...
Chhlohlolorororoquininineeeee ee isisisiss a aaaa aantaaantntntimimimimmalaaarariaararariaiaiaia a a aagegegeeentntntntnt ofof 4ofofof 4444-a-a-a-amiamimiminonnonono q q q q qqquuuiuiuiunon liliinenene gro nrosidddalalal t t to oo alallall l kikikikkindndnd ondnd o o of off ffPlasPlPlPlPlasasasasmomomomomoddiumdidddumumumum a aaaandnd andndnd a alsaalslslslso o go g g gamgamamamametmmettosososidididalalal t t toP.
ae. The drdrdrugugug wwwerereeree dedededededeelivelilililivevevev rrerer d ddddtotototo d ddddigigiigegessttitititiveveveveve vavavavavaaaccucucuc ololeee tototo i iinhnhnhibibi it prot alciparum.
P. falciparum resessisisistatatancncnceee tototo ant aantntimimimalalalararariaiaia drdrdrugs are serous p
were given properly. P, falciparum chloroquine resistance in vitro are maximum concentration of chloroquine that have capable to kill 50% parasite (IC50). The mechanism of chloroquine reistance based on fault of chloroquine inhibited heme polymerase synthesis. P. falciparum continue to produce hematin to be hemozoin polymer. The strain of P, falciparum chloroquine resistance succed reducing chloroquine accumulation in digestive vacuole. The detail of resistance mechanisms remains unclear.
The data from all region of world showed 12 point mutations of pfcrt gene related to chloroquine resistance. The mutation could be cause chloroquine induction and spreading of spontaneous mutation from others area. Twelve points mutation caused alteration of amino acid codons: cysteine to serine in codon 72 (C72S), methionine to isoleucine in codon 74 (M74I), asparagine to glutamic acid or aspartic acid in codon 76 (N75E or N75D). lysine to threonine in codon 76 (K76T), histidine to leucine or glutamine in codon 97 (H97L or H97Q), alanine to threonine in codon 144 (A144T), lecine to tyrosine in codon 160 (L160Y), alanine to serine in codon 220 (A220S), glutamine to glutamic acid in codon 271 (Q271E), asparagine to serine in codon 326 (N326S), or aspartic acid (N326D), isoleucine to threonine or leucine in codon 356 (I356T or I356L)), and arginine to isoleucine or threonine in codon 371 (R371I or R371T). The point mutation distribution patterns related to geographic and demographic are still quenstionable.
The researcher was interested in studying about the point mutation patterns in three differences geographic and demographic areas in South Sumatera. The aim of this research is to identify and describe point mutation the distribution of pattern of pfcrt gene based on topography (one aspect of geography) and demography differences including remote area, tourism area and highly population mobility area. This molecular epidemiology with descriptive exploratoric approach of children 1-18 years old patients from 3 endemic malaria area in South Sumatera.Tanjung Lengkayap OKU Induk is a remote area, Banding Agung OKU Selatan is a tourism area and Belitang OKU Timur is an area with highly population mobility. The steps of study were spleen rate survey, clinical malaria diagnosis, subject recruitment and blood specimen collection, microscopic
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Desertasi Mutasi titik gen pfcrt pada penderita .... Rosiana A. Marbun on and spreading of spopopontntntanananeoeoeoususus m m mutututatatatioioion from others area. T
on caused alteeerararatititiononon ooof f f amamaminnno oo acacacididd c ccodododons:onons:s: c c cysysysteteteine to serine , methioninininenene tttoo o isisisolololeuuuciciciinnnenenenene i iiiinn n n ncccodcccododododononononon 7 7 7777444 4 4 4(M(((M(M(M(M747474I)I)I), asasaspapaparararagig ne to g artic acaccididid i i in n n codoccododoooon n nnn 767776767676 ( ( (N7((N7NN7N7N75E55E5E5E5E o ooor or N7NNN7N7N7N75D5D5D5D5D).).).).). l ly l llyysysysy ine inininininin tototo t t thrhrhreoeoeonine
), hiistststidididine e etototoo looo l llleueueueueucicciciccnennenne ororororr grrr glllululullluutamtatatatammmmininininineeee einininii c codcododododonon 9onononn 9 9997 77 77 (H(((H((H(H979797LL L ororor H9 oniniinenene iiin nn cococoodooododododod n n nn n 1414414141444 44 (A(AAAAA1141144T141444T4T4T4T),),),),), llecllececececiiiiinenenenene t to tttoo oo tytytytytyrorosirororosisisinenenenee iniinininnnn codccododononon 1
to to
to ssserererinininee e inininininn c c codccodododoon oonnn n 222222222200000 (A(A(A(A22A22220S0S0S)SS))), ggggglul taaamimimimminenenenene ttott glglglglglutututututamamamamamicmmicic a a acicicd dd
E)),), aaaspspsparararagaagggininininine eeetoto stototo s seseeeeriririneririnenenene i iiin n nn cccodon 32323232326 6666 (NN32NNN323232326S6S6S6S6S),)), o)), o oorrrrrrraasspspspararartititic caaac inenene tttooo thththreeeeeonononononinine ininneeee or leleleleucuu ine inin cinccccodododododonon 3ononon 3 3 3356565666 ((I3(I3I3I33356565656T6T oTTT o ooorrrr rrI33565656L)L)L)))), euccininine e ororor tthhhrhrhrhreooonineninininenene e ininininin c ccodddddononononon 371 37373771 1 1 (R(R(R(R(R373737771I11I1I1I oI o oor orr rr R3R3R3R3R37171T)717171T)T). ThThThee e po utionnn p p patatatteteternrnrnns sss ss relarerererelalalal teted teteteddd d tototototo ggeggeeeeogeogooograraphrararaphphphphicicc acc aaaandndndndn dddemememogogograraraphi
onable.e.e
The researrrchchcher werer wwasasas i intintnterereresesestetetetetededd d dinin snnn sstututututuuuddyddyddydyinining g gaboubououtt t thththe e e pppoint muta e differences geoeoeogrgrgrapapaphihihic cc aana d d demdedemogmoggrararaphicphphicic a a arerereas in South Su
xxxi
examination, chloroquine in vitro test, chloroquine in vivo test, DNA isolation, PCR and sequencing.. The results showed that 3 areas were hypoendemic malaria areas based on the average of spleen rate less than 10%. About 562 blood samples based on clinical malaria diagnosis were defined with microscopic examination and got 171 (30.4%) samples with falciparum malaria. The group of 5-12 years old were 94 (55%) patients. The history of rigor was highly found in Banding Agung (62.7%). Palmar pale dominant found in Tanjung Lengkayap (39.3%), also spleen enlargement about 20.2%. The history of anti malaria drug therapy 41.7% found in Tanjung Lengkayap. The temperature examination results > 37.5oC 58.3% found in Belitang. Twenty five sample tried to in vitro test three times but the parasite did not growed, so the others 146 samples conducted to in vivo test. The results of in vivo showed that 44 (30.1%) samples were resintance to chloroquine. The 168 (98.5%) samples can be isolated of DNA for PCR an sequencing. The point mutation were found in codons: M74I, N75L, K76T, H97F, A220S, and I356L. one hundred percent of codons 74, 75, 76, and 356 were mutated. The mutation of codon 97 in Tanjung Lengkayap, Banding Agung, and Belitang 44%, 46.9%, and 60% respectively. The mutation of codon 220 in Tanjung Lengkayap, Banding Agung 40.5%, 46.9%, and 60% respectively. The result showed that in three areas, the pattern of mutation were the same in codons 74, 75, 76, and 356 and getting variation at codons 97 and 220. The point mutation of codon 97 and 220 were dominant in Belitang. This study showed that the mutation was multiple point mutation. The new finding or local mutation were found at codons N75L and H97F. Until now never were they find.
Desertasi Mutasi titik gen pfcrt pada penderita .... Rosiana A. Marbun found in Belitang. Twewewentntnty y y fififiveveve s ssamamamplplple e e trtrtried to in vitro test th
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