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Open Access 01-12-2015 | Research

Identification of large variation in pfcrt, pfmdr-1 and pfubp-1 markers in Plasmodium falciparum isolates from Ethiopia and Tanzania

Authors: Lemu Golassa, Erasmus Kamugisha, Deus S Ishengoma, Vito Baraka, Alex Shayo, Frederick N Baliraine, Nizar Enweji, Berhanu Erko, Abraham Aseffa, Angel Choy, Göte Swedberg

Published in: Malaria Journal | Issue 1/2015

Abstract

Background

Plasmodium falciparum resistance to anti-malarials is a major drawback in effective malaria control and elimination globally. Artemisinin-combination therapy (ACT) is currently the key first-line treatment for uncomplicated falciparum malaria. Plasmodium falciparum genetic signatures at pfmdr-1, pfcrt, and pfubp-1 loci are known to modulate in vivo and in vitro parasite response to ACT. The objective of this study was to assess the distribution of these resistance gene markers in isolates collected from different malaria transmission intensity in Ethiopia and Tanzania.

Methods

Plasmodium falciparum clinical isolates were collected from different regions of Ethiopia and Tanzania. Genetic polymorphisms in the genes pfcrt, pfmdr-1 and pfubp-1 were analysed by PCR and sequencing. Frequencies of the different alleles in the three genes were compared within and between regions, and between the two countries.

Results

The majority of the isolates from Ethiopia were mutant for the pfcrt 76 and wild-type for pfmdr-1 86. In contrast, the majority of the Tanzanian samples were wild-type for both pfcrt and pfmdr-1 loci. Analysis of a variable linker region in pfmdr-1 showed substantial variation in isolates from Tanzania as compared to Ethiopian isolates that had minimal variation. Direct sequencing of the pfubp-1 region showed that 92.8% (26/28) of the Ethiopian isolates had identical genome sequence with the wild type reference P. falciparum strain 3D7. Of 42 isolates from Tanzania, only 13 (30.9%) had identical genome sequences with 3D7. In the Tanzanian samples, 10 variant haplotypes were identified.

Conclusion

The majority of Ethiopian isolates carried the main marker for chloroquine (CQ) resistance, while the majority of the samples from Tanzania carried markers for CQ susceptibility. Polymorphic genes showed substantially more variation in Tanzanian isolates. The low variability in the polymorphic region of pfmdr-1 in Ethiopia may be a consequence of low transmission intensity as compared to high transmission intensity and large variations in Tanzania.

Sisowath C, Ferreira PE, Bustamante LY, Dahlstrom S, Mårtensson A, Bjorkman A et al (2007) The role of pfmdr1 in Plasmodium falciparum tolerance to artemether-lumefantrine in Africa. Trop Med Int Health 12:736–742PubMedCrossRef

Humphreys GS, Merinopoulos I, Ahmed J, Whitty CJ, Mutabingwa TK, Sutherland CJ et al (2007) Amodiaquine and artemether-lumefantrine select distinct alleles of the Plasmodium falciparum mdr1 gene in Tanzanian children treated for uncomplicated malaria. Antimicrob Agents Chemother 51:991–997PubMedCentralPubMedCrossRef

Sisowath C, Petersen I, Veiga MI, Mårtensson A, Premji Z, Bjorkman A et al (2009) In vivo selection of Plasmodium falciparum parasites carrying the chloroquine-susceptible pfcrt K76 allele after treatment with artemether-lumefantrine in Africa. J Infect Dis 199:750–757PubMedCentralPubMedCrossRef

Conrad MD, LeClair N, Arinaitwe E, Wanzira H, Kakuru A, Bigira V et al (2014) Comparative impacts over 5 years of artemisinin-based combination therapies on Plasmodium falciparum polymorphisms that modulate drug sensitivity in Ugandan children. J Infect Dis 210:344–353PubMedCrossRef

Duraisingh MT, von Seidlein LV, Jepson A, Jones P, Sambou I, Pinder M et al (2000) Linkage disequilibrium between two chromosomally distinct loci associated with increased resistance to chloroquine in Plasmodium falciparum. Parasitology 121:1–7PubMedCrossRef

Duraisingh MT, Cowman AF (2005) Contribution of the pfmdr1 gene to antimalarial drug-resistance. Acta Trop 94:181–190PubMedCrossRef

Suwanarusk R, Russell B, Chavchich M, Chalfein F, Kenangalem E, Kosaisavee V et al (2007) Chloroquine resistant Plasmodium vivax: in vitro characterisation and association with molecular polymorphisms. PLoS One 2:e1089PubMedCentralPubMedCrossRef

Mwai L, Ochong E, Abdirahman A, Kiara SM, Ward S, Kokwaro G et al (2009) Chloroquine resistance before and after its withdrawal in Kenya. Malar J 8:106PubMedCentralPubMedCrossRef

Baliraine FN, Rosenthal PJ (2011) Prolonged selection of pfmdr1 polymorphisms after treatment of falciparum malaria with artemether-lumefantrine in Uganda. J Infect Dis 204:1120–1124PubMedCentralPubMedCrossRef

10.

Baraka V, Tinto H, Valea I, Fitzhenry R, Delgado-Ratto C, Mbonye MK et al (2015) In vivo selection of Plasmodium falciparum Pfcrt and Pfmdr1 variants by artemether-lumefantrine and dihydroartemisinin-piperaquine in Burkina Faso. Antimicrob Agents Chemother 59:734–737PubMedCentralPubMedCrossRef

11.

Djimde AA, Fofana B, Sagara I, Sidibe B, Toure S, Dembele D et al (2008) Efficacy, safety, and selection of molecular markers of drug resistance by two ACTs in Mali. Am J Trop Med Hyg 78:455–461PubMed

12.

Legrand E, Yrinesi J, Ekala MT, Peneau J, Volney B, Berger F et al (2012) Discordant temporal evolution of Pfcrt and Pfmdr1 genotypes and Plasmodium falciparum in vitro drug susceptibility to 4-aminoquinolines after drug policy change in French Guiana. Antimicrob Agents Chemother 56:1382–1389PubMedCentralPubMedCrossRef

13.

Wang X, Mu J, Li G, Chen P, Guo X, Fu L et al (2005) Decreased prevalence of the Plasmodium falciparum chloroquine resistance transporter 76T marker associated with cessation of chloroquine use against P. falciparum malaria in Hainan, People’s Republic of China. Am J Trop Med Hyg 72:410–414PubMed

14.

Laufer MK, Takala-Harrison S, Dzinjalamala FK, Stine OC, Taylor TE, Plowe CV (2010) Return of chloroquine-susceptible falciparum malaria in Malawi was a reexpansion of diverse susceptible parasites. J Infect Dis 202:801–808PubMedCentralPubMedCrossRef

15.

Kublin JG, Cortese JF, Njunju EM, Mukadam RA, Wirima JJ, Kazembe PN et al (2003) Reemergence of chloroquine-sensitive Plasmodium falciparum malaria after cessation of chloroquine use in Malawi. J Infect Dis 187:1870–1875PubMedCrossRef

16.

Baliraine FN, Nsobya SL, Achan J, Tibenderana JK, Talisuna AO, Greenhouse B et al (2011) Limited ability of Plasmodium falciparum pfcrt, pfmdr1, and pfnhe1 polymorphisms to predict quinine in vitro sensitivity or clinical effectiveness in Uganda. Antimicrob Agents Chemother 55:615–622PubMedCentralPubMedCrossRef

17.

Happi CT, Gbotosho GO, Folarin OA, Sowunmi A, Hudson T, O’Neil M et al (2009) Selection of Plasmodium falciparum multidrug resistance gene 1 alleles in asexual stages and gametocytes by artemether-lumefantrine in Nigerian children with uncomplicated falciparum malaria. Antimicrob Agents Chemother 53:888–895PubMedCentralPubMedCrossRef

18.

Frosch AE, Laufer MK, Mathanga DP, Takala-Harrison S, Skarbinski J, Claassen CW et al (2014) Return of widespread chloroquine-sensitive Plasmodium falciparum to Malawi. J Infect Dis 210:1110–1114PubMedCrossRef

19.

Aregawi MW, Ali AS, Al-mafazy AW, Molteni F, Katikiti S, Warsame M et al (2011) Reductions in malaria and anaemia case and death burden at hospitals following scale-up of malaria control in Zanzibar, 1999–2008. Malar J 10:46PubMedCentralPubMedCrossRef

20.

Karema C, Aregawi MW, Rukundo A, Kabayiza A, Mulindahabi M, Fall IS et al (2012) Trends in malaria cases, hospital admissions and deaths following scale-up of anti-malarial interventions, 2000–2010, Rwanda. Malar J 11:236PubMedCentralPubMedCrossRef

21.

Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM (2008) Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med 359:2619–2620PubMedCrossRef

22.

Dondorp AM, Fairhurst RM, Slutsker L, Macarthur JR, Breman JG, Guerin PJ et al (2011) The threat of artemisinin-resistant malaria. N Engl J Med 365:1073–1075PubMedCentralPubMedCrossRef

23.

Phyo AP, Nkhoma S, Stepniewska K, Ashley EA, Nair S, McGready R et al (2012) Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. Lancet 379:1960–1966PubMedCentralPubMedCrossRef

24.

Kublin JG, Dzinjalamala FK, Kamwendo DD, Malkin EM, Cortese JF, Martino LM et al (2002) Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis 185:380–388PubMedCrossRef

25.

Ariey F, Fandeur T, Durand R, Randrianarivelojosia M, Jambou R, Legrand E et al (2006) Invasion of Africa by a single pfcrt allele of South East Asian type. Malar J 5:34PubMedCentralPubMedCrossRef

26.

Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J et al (2009) Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 361:455–467PubMedCentralPubMedCrossRef

27.

Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N et al (2014) A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505:50–55PubMedCrossRef

28.

Kyaw MP, Nyunt MH, Chit K, Aye MM, Aye KH, Aye MM et al (2013) Reduced susceptibility of Plasmodium falciparum to artesunate in southern Myanmar. PLoS ONE 8:e57689PubMedCentralPubMedCrossRef

29.

Takala-Harrison S, Jacob CG, Arze C, Cummings MP, Silva JC, Dondorp AM et al (2015) Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia. J Infect Dis 211:670–679PubMedCrossRef

30.

Kamau E, Campino S, Amenga-Etego L, Drury E, Ishengoma D, Johnson K et al (2015) K13-propeller polymorphisms in Plasmodium falciparum parasites from sub-Saharan Africa. J Infect Dis 211:1352–1355PubMed

31.

Hunt P, Afonso A, Creasey A, Culleton R, Sidhu AB, Logan J et al (2007) Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites. Mol Microbiol 65:27–40PubMedCentralPubMedCrossRef

32.

Hunt P, Martinelli A, Modrzynska K, Borges S, Creasey A, Rodrigues L et al (2010) Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites. BMC Genom 11:499CrossRef

33.

Borrmann S, Straimer J, Mwai L, Abdi A, Rippert A, Okombo J et al (2013) Genome-wide screen identifies new candidate genes associated with artemisinin susceptibility in Plasmodium falciparum in Kenya. Sci Rep 3:3318PubMedCentralPubMedCrossRef

34.

Henriques G, Hallett RL, Beshir KB, Gadalla NB, Johnson RE, Burrow R et al (2014) Directional selection at the pfmdr1, pfcrt, pfubp1, and pfap2mu loci of Plasmodium falciparum in Kenyan children treated With ACT. J Infect Dis 210:2001–2008PubMedCentralPubMedCrossRef

35.

Miotto O, Almagro-Garcia J, Manske M, Macinnis B, Campino S, Rockett KA et al (2013) Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia. Nat Genet 45:648–655PubMedCrossRef

36.

Gadalla NB, Adam I, Elzaki SE, Bashir S, Mukhtar I, Oguike M et al (2011) Increased pfmdr1 copy number and sequence polymorphisms in Plasmodium falciparum isolates from Sudanese malaria patients treated with artemether-lumefantrine. Antimicrob Agents Chemother 55:5408–5411PubMedCentralPubMedCrossRef

37.

Kiwuwa MS, Byarugaba J, Wahlgren M, Kironde F (2013) Detection of copy number variation and single nucleotide polymorphisms in genes involved in drug resistance and other phenotypic traits in P. falciparum clinical isolates collected from Uganda. Acta Trop 125:269–275PubMedCrossRef

38.

Abeku TA, van Oortmarssen GJ, Borsboom G, de Vlas SJ, Habbema JD (2003) Spatial and temporal variations of malaria epidemic risk in Ethiopia: factors involved and implications. Acta Trop 87:331–340PubMedCrossRef

39.

Mulligan JA, Mandike R, Palmer N, Williams H, Abdulla S, Bloland P et al (2006) The costs of changing national policy: lessons from malaria treatment policy guidelines in Tanzania. Trop Med Int Health 11:452–461PubMedCrossRef

40.

McKenzie FE, Prudhomme WA, Magill AJ, Forney JR, Permpanich B, Lucas C et al (2005) White blood cell counts and malaria. J Infect Dis 192:323–330PubMedCentralPubMedCrossRef

41.

Plowe CV, Djimde A, Bouare M, Doumbo O, Wellems TE (1995) Pyrimethamine and proguanil resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for surveillance in Africa. Am J Trop Med Hyg 52:565–568PubMed

42.

Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y et al (2001) A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344:257–263PubMedCrossRef

43.

Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT et al (2000) Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6:861–871PubMedCentralPubMedCrossRef

44.

Mehlotra RK, Mattera G, Bockarie MJ, Maguire JD, Baird JK, Sharma YD et al (2008) Discordant patterns of genetic variation at two chloroquine resistance loci in worldwide populations of the malaria parasite Plasmodium falciparum. Antimicrob Agents Chemother 52:2212–2222PubMedCentralPubMedCrossRef

45.

Mekonnen SK, Aseffa A, Berhe N, Teklehaymanot T, Clouse RM, Gebru T et al (2014) Return of chloroquine-sensitive Plasmodium falciparum parasites and emergence of chloroquine-resistant Plasmodium vivax in Ethiopia. Malar J 13:244PubMedCentralPubMedCrossRef

46.

Pillai DR, Labbe AC, Vanisaveth V, Hongvangthong B, Pomphida S, Inkathone S et al (2001) Plasmodium falciparum malaria in Laos: chloroquine treatment outcome and predictive value of molecular markers. J Infect Dis 183:789–795PubMedCrossRef

47.

Abdel-Muhsin AM, Mackinnon MJ, Ali E, el Nassir KA, Suleiman S, Ahmed S et al (2004) Evolution of drug-resistance genes in Plasmodium falciparum in an area of seasonal malaria transmission in Eastern Sudan. J Infect Dis 189:1239–1244PubMedCrossRef

48.

Kamugisha E, Bujila I, Lahdo M, Pello-Esso S, Minde M, Kongola G et al (2012) Large differences in prevalence of Pfcrt and Pfmdr1 mutations between Mwanza, Tanzania and Iganga, Uganda-a reflection of differences in policies regarding withdrawal of chloroquine? Acta Trop 121:148–151PubMedCrossRef

49.

Laufer MK, Plowe CV (2004) Withdrawing antimalarial drugs: impact on parasite resistance and implications for malaria treatment policies. Drug Resist Updat 7:279–288PubMedCrossRef

50.

Ochola LI, Tetteh KK, Stewart LB, Riitho V, Marsh K, Conway DJ (2010) Allele frequency-based and polymorphism-versus-divergence indices of balancing selection in a new filtered set of polymorphic genes in Plasmodium falciparum. Mol Biol Evol 27:2344–2351PubMedCentralPubMedCrossRef

51.

Nsobya SL, Kiggundu M, Nanyunja S, Joloba M, Greenhouse B, Rosenthal PJ (2010) In vitro sensitivities of Plasmodium falciparum to different antimalarial drugs in Uganda. Antimicrob Agents Chemother 54:1200–1206PubMedCentralPubMedCrossRef

52.

Setthaudom C, Tan-ariya P, Sitthichot N, Khositnithikul R, Suwandittakul N, Leelayoova S et al (2011) Role of Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes on in vitro chloroquine resistance in isolates of Plasmodium falciparum from Thailand. Am J Trop Med Hyg 85:606–611PubMedCentralPubMedCrossRef

Title: Identification of large variation in pfcrt, pfmdr-1 and pfubp-1 markers in Plasmodium falciparum isolates from Ethiopia and Tanzania
Authors: Lemu Golassa
Erasmus Kamugisha
Deus S Ishengoma
Vito Baraka
Alex Shayo
Frederick N Baliraine
Nizar Enweji
Berhanu Erko
Abraham Aseffa
Angel Choy
Göte Swedberg
Publication date: 01-12-2015
Publisher: BioMed Central
Published in: Malaria Journal / Issue 1/2015
Electronic ISSN: 1475-2875
DOI: https://doi.org/10.1186/s12936-015-0783-3

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Identification of large variation in pfcrt, pfmdr-1 and pfubp-1 markers in Plasmodium falciparum isolates from Ethiopia and Tanzania

Abstract

Background

Methods

Results

Conclusion

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