Molecular analysis demonstrates high prevalence of chloroquine resistance but no evidence of artemisinin resistance in Plasmodium falciparum in the Chittagong Hill Tracts of Bangladesh

Mohammad Shafiul Alam, Benedikt Ley, Maisha Khair Nima, Fatema Tuj Johora, Mohammad Enayet Hossain, Kamala Thriemer, Sarah Auburn, Jutta Marfurt, Ric N Price, Wasif A Khan, Mohammad Shafiul Alam, Benedikt Ley, Maisha Khair Nima, Fatema Tuj Johora, Mohammad Enayet Hossain, Kamala Thriemer, Sarah Auburn, Jutta Marfurt, Ric N Price, Wasif A Khan

Abstract

Background: Artemisinin resistance is present in the Greater Mekong region and poses a significant threat for current anti-malarial treatment guidelines in Bangladesh. The aim of this molecular study was to assess the current status of drug resistance in the Chittagong Hill Tracts of Bangladesh near the Myanmar border.

Methods: Samples were obtained from patients enrolled into a Clinical Trial (NCT02389374) conducted in Alikadam, Bandarban between August 2014 and January 2015. Plasmodium falciparum infections were confirmed by PCR and all P. falciparum positive isolates genotyped for the pfcrt K76T and pfmdr1 N86Y markers. The propeller region of the kelch 13 (k13) gene was sequenced from isolates from patients with delayed parasite clearance.

Results: In total, 130 P. falciparum isolates were available for analysis. The pfcrt mutation K76T, associated with chloroquine resistance was found in 81.5% (106/130) of cases and the pfmdr1 mutation N86Y in 13.9% (18/130) cases. No single nucleotide polymorphisms were observed in the k13 propeller region.

Conclusion: This study provides molecular evidence for the ongoing presence of chloroquine resistant P. falciparum in Bangladesh, but no evidence of mutations in the k13 propeller domain associated with artemisinin resistance. Monitoring for artemisinin susceptibility in Bangladesh is needed to ensure early detection and containment emerging anti-malarial resistance.

Keywords: Antimalarial drug resistance; Artemisinin; Bangladesh; K13 propeller; Molecular markers; Plasmodium falciparum; Single nucleotide polymorphism; pfcrt; pfmdr1.

References

    1. World Health Organization . Global technical strategy for Malaria 2016–2030. Geneva: World Health Organization; 2015.
    1. Haque U, Overgaard HJ, Clements AC, Norris DE, Islam N, Karim J, et al. Malaria burden and control in Bangladesh and prospects for elimination: an epidemiological and economic assessment. Lancet Glob Health. 2014;2:e98–e105. doi: 10.1016/S2214-109X(13)70176-1.
    1. Haque U, Ahmed SM, Hossain S, Huda M, Hossain A, Alam MS, et al. Malaria prevalence in endemic districts of Bangladesh. PLoS ONE. 2009;4:e6737. doi: 10.1371/journal.pone.0006737.
    1. Starzengruber P, Fuehrer HP, Ley B, Thriemer K, Swoboda P, Habler VE, et al. High prevalence of asymptomatic malaria in south-eastern Bangladesh. Malar J. 2014;13:16. doi: 10.1186/1475-2875-13-16.
    1. Maude RJ, Hasan MU, Hossain MA, Sayeed AA, Paul SK, Rahman W, et al. Temporal trends in severe malaria in Chittagong, Bangladesh. Malar J. 2012;11:323. doi: 10.1186/1475-2875-11-323.
    1. Alam MS, Chakma S, Khan WA, Glass GE, Mohon AN, Elahi R, et al. Diversity of anopheline species and their Plasmodium infection status in rural Bandarban, Bangladesh. Parasit Vectors. 2012;5:150. doi: 10.1186/1756-3305-5-150.
    1. Alam MS, Khan MGM, Chaudhury N, Deloer S, Nazib F, Bangali AM, et al. Prevalence of anopheline species and their Plasmodium infection status in epidemic-prone border areas of Bangladesh. Malar J. 2010;9:15. doi: 10.1186/1475-2875-9-15.
    1. Al-Amin HM, Elahi R, Mohon AN, Kafi MAH, Chakma S, Lord JS, et al. Role of underappreciated vectors in malaria transmission in an endemic region of Bangladesh-India border. Parasit Vectors. 2015;8:195. doi: 10.1186/s13071-015-0803-8.
    1. DGHS B. Strategic plan for malaria control Programme Bangladesh; 2008.
    1. Marma AS, Mita T, Eto H, Tsukahara T, Sarker S, Endo H. High prevalence of sulfadoxine/pyrimethamine resistance alleles in Plasmodium falciparum parasites from Bangladesh. Parasitol Int. 2010;59:178–182. doi: 10.1016/j.parint.2010.01.003.
    1. Woodrow CJ, White NJ. The clinical impact of artemisinin resistance in Southeast Asia and the potential for future spread. FEMS Microbiol Rev. 2017;41:34–48. doi: 10.1093/femsre/fuw037.
    1. Ibraheem ZO, Abd Majid R, Noor SM, Sedik HM, Basir R. Role of different Pfcrt and Pfmdr-1 mutations in conferring resistance to antimalaria drugs in Plasmodium falciparum. Malar Res Treat. 2014;2014:950424.
    1. Ecker A, Lehane AM, Clain J, Fidock DA. PfCRT and its role in antimalarial drug resistance. Trends Parasitol. 2012;28:504–514. doi: 10.1016/j.pt.2012.08.002.
    1. Duraisingh MT, Cowman AF. Contribution of the pfmdr1 gene to antimalarial drug-resistance. Acta Trop. 2005;94:181–190. doi: 10.1016/j.actatropica.2005.04.008.
    1. Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature. 2000;403:906–909. doi: 10.1038/35002615.
    1. Eyase FL, Akala HM, Ingasia L, Cheruiyot A, Omondi A, Okudo C, et al. The role of Pfmdr1 and Pfcrt in changing chloroquine, amodiaquine, mefloquine and lumefantrine susceptibility in western-Kenya P. falciparum samples during 2008–2011. PLoS ONE. 2013;8:e64299. doi: 10.1371/journal.pone.0064299.
    1. Price RN, Uhlemann AC, Brockman A, McGready R, Ashley E, Phaipun L, et al. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet. 2004;364:438–447. doi: 10.1016/S0140-6736(04)16767-6.
    1. Sidhu ABS, Uhlemann AC, Valderramos SG, Valderramos JC, Krishna S, Fidock DA. Decreasing pfmdr1 copy number in Plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J Infect Dis. 2006;194:528–535. doi: 10.1086/507115.
    1. Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM. Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med. 2008;359:2619–2620. doi: 10.1056/NEJMc0805011.
    1. Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, et al. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2009;361:455–467. doi: 10.1056/NEJMoa0808859.
    1. Vijaykadga S, Rojanawatsirivej C, Cholpol S, Phoungmanee D, Nakavej A, Wongsrichanalai C. In vivo sensitivity monitoring of mefloquine monotherapy and artesunate–mefloquine combinations for the treatment of uncomplicated falciparum malaria in Thailand in 2003. Trop Med Int Health. 2006;11:211–219. doi: 10.1111/j.1365-3156.2005.01557.x.
    1. Jambou R, Legrand E, Niang M, Khim N, Lim P, Volney B, et al. Resistance of Plasmodium falciparum field isolates to in vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet. 2005;366:1960–1963. doi: 10.1016/S0140-6736(05)67787-2.
    1. Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2014;505:50–55. doi: 10.1038/nature12876.
    1. Miotto O, Amato R, Ashley EA, MacInnis B, Almagro-Garcia J, Amaratunga C, et al. Genetic architecture of artemisinin-resistant Plasmodium falciparum. Nat Genet. 2015;47:226–234. doi: 10.1038/ng.3189.
    1. van den Broek IV, van der Wardt S, Talukder L, Chakma S, Brockman A, Nair S, et al. Drug resistance in Plasmodium falciparum from the Chittagong Hill Tracts, Bangladesh. Trop Med Int Health. 2004;9:680–687. doi: 10.1111/j.1365-3156.2004.01249.x.
    1. Kawai A, Arita N, Matsumoto Y, Kawabata M, Chowdhury MS, Saito-Ito A. Efficacy of chloroquine plus primaquine treatment and pfcrt mutation in uncomplicated falciparum malaria patients in Rangamati,Bangladesh. Parasitol Int. 2011;60:341–346. doi: 10.1016/j.parint.2011.05.007.
    1. Akter J, Thriemer K, Khan WA, Sullivan DJ, Noedl H, Haque R. Genotyping of Plasmodium falciparum using antigenic polymorphic markers and to study anti-malarial drug resistance markers in malaria endemic areas of Bangladesh. Malar J. 2012;11:386. doi: 10.1186/1475-2875-11-386.
    1. Srimuang K, Miotto O, Lim P, Fairhurst RM, Kwiatkowski DP, Woodrow CJ, et al. Analysis of anti-malarial resistance markers in pfmdr1 and pfcrt across Southeast Asia in the tracking resistance to artemisinin collaboration. Malar J. 2016;15:541. doi: 10.1186/s12936-016-1598-6.
    1. Mohon AN, Alam MS, Bayih AG, Folefoc A, Shahinas D, Haque R, et al. Mutations in Plasmodium falciparum K13 propeller gene from Bangladesh (2009–2013) Malar J. 2014;13:431. doi: 10.1186/1475-2875-13-431.
    1. Menard D, Khim N, Beghain J, Adegnika AA, Shafiul-Alam M, Amodu O, et al. A worldwide map of Plasmodium falciparum K13-propeller polymorphisms. N Engl J Med. 2016;374:2453–2464. doi: 10.1056/NEJMoa1513137.
    1. Nyunt MH, Hlaing T, Oo HW, Tin-Oo LLK, Phway HP, Wang B, et al. Molecular assessment of artemisinin resistance markers, polymorphisms in the K13 propeller, and a multidrug-resistance gene in the eastern and western border areas of Myanmar. Clin Infect Dis. 2014;60:1208–1215. doi: 10.1093/cid/ciu1160.
    1. Tun KM, Imwong M, Lwin KM, Win AA, Hlaing TM, Hlaing T, et al. Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker. Lancet Infect Dis. 2015;15:415–421. doi: 10.1016/S1473-3099(15)70032-0.
    1. Mishra N, Bharti RS, Mallick P, Singh OP, Srivastava B, Rana R, et al. Emerging polymorphisms in falciparum kelch 13 gene in Northeastern region of India. Malar J. 2016;15:583. doi: 10.1186/s12936-016-1636-4.
    1. Ley B, Alam MS, Thriemer K, Hossain MS, Kibria MG, Auburn S, et al. G6PD deficiency and antimalarial efficacy for uncomplicated malaria in Bangladesh: a prospective observational study. PLoS ONE. 2016;11:e0154015. doi: 10.1371/journal.pone.0154015.
    1. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosario V, et al. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol. 1993;61:315–320. doi: 10.1016/0166-6851(93)90077-B.
    1. Veiga MI, Ferreira PE, Bjorkman A, Gil JP. Multiplex PCR-RFLP methods for pfcrt, pfmdr1 and pfdhfr mutations in Plasmodium falciparum. Mol Cell Probes. 2006;20:100–104. doi: 10.1016/j.mcp.2005.10.003.
    1. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011;7:539. doi: 10.1038/msb.2011.75.
    1. Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, et al. A new bioinformatics analysis tools framework at EMBL–EBI. Nucleic Acids Res. 2010;38:W695–W699. doi: 10.1093/nar/gkq313.
    1. Alam MS, Elahi R, Mohon AN, Al-Amin HM, Kibria MG, Khan WA, et al. Plasmodium falciparum genetic diversity in Bangladesh does not suggest a hypoendemic population structure. Am J Trop Med Hyg. 2016;94:1245–1250. doi: 10.4269/ajtmh.15-0446.
    1. Snounou G, Zhu X, Siripoon N, Jarra W, Thaithong S, Brown KN, et al. Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand. Trans R Soc Trop Med Hyg. 1999;93:369–374. doi: 10.1016/S0035-9203(99)90120-7.
    1. Noedl H, Faiz M, Yunus E, Rahman M, Hossain M, Samad R, et al. Drug-resistant malaria in Bangladesh: an in vitro assessment. Am J Trop Med Hyg. 2003;68:140–142.
    1. Thriemer K, Haque R, Wagatsuma Y, Salam MA, Akther S, Attlmayr B, et al. Therapeutic efficacy of quinine plus sulfadoxine–pyremethamine for the treatment of uncomplicated falciparum malaria in Bangladesh. Am J Trop Med Hyg. 2006;75:645–649.
    1. Laufer MK, Thesing PC, Eddington ND, Masonga R, Dzinjalamala FK, Takala SL, et al. Return of chloroquine antimalarial efficacy in Malawi. N Engl J Med. 2006;355:1959–1966. doi: 10.1056/NEJMoa062032.
    1. Mohammed A, Ndaro A, Kalinga A, Manjurano A, Mosha JF, Mosha DF, et al. Trends in chloroquine resistance marker, Pfcrt-K76T mutation ten years after chloroquine withdrawal in Tanzania. Malar J. 2013;12:415. doi: 10.1186/1475-2875-12-415.
    1. Mwanza S, Joshi S, Nambozi M, Chileshe J, Malunga P, Kabuya JB, et al. The return of chloroquine-susceptible Plasmodium falciparum malaria in Zambia. Malar J. 2016;15:584. doi: 10.1186/s12936-016-1637-3.
    1. Huang B, Wang Q, Deng C, Wang J, Yang T, Huang S, et al. Prevalence of crt and mdr-1 mutations in Plasmodium falciparum isolates from Grande Comore island after withdrawal of chloroquine. Malar J. 2016;15:414. doi: 10.1186/s12936-016-1474-4.
    1. Frosch AE, Venkatesan M, Laufer MK. Patterns of chloroquine use and resistance in sub-Saharan Africa: a systematic review of household survey and molecular data. Malar J. 2011;10:116. doi: 10.1186/1475-2875-10-116.
    1. Gharbi M, Flegg J, Hubert V, Kendjo E, Metcalf J, Guerin PJ, et al. Longitudinal study assessing the return of chloroquine susceptibility of Plasmodiun falciparum isolates from travelers returning from West Africa. Malar J. 2012;11:P38. doi: 10.1186/1475-2875-11-S1-P38.
    1. Duru V, Witkowski B, Menard D. Plasmodium falciparum resistance to artemisinin derivatives and piperaquine: a major challenge for malaria elimination in Cambodia. Am J Trop Med Hyg. 2016;95:1228–1238. doi: 10.4269/ajtmh.16-0234.
    1. Chatterjee M, Ganguly S, Saha P, Guha SK, Basu N, Bera DK, et al. Polymorphisms in Pfcrt and Pfmdr-1 genes after five years withdrawal of chloroquine for the treatment of Plasmodium falciparum malaria in West Bengal, India. Infect Genet Evol. 2016;44:281–285. doi: 10.1016/j.meegid.2016.07.021.
    1. Takahashi N, Tanabe K, Tsukahara T, Dzodzomenyo M, Dysoley L, Khamlome B, et al. Large-scale survey for novel genotypes of Plasmodium falciparum chloroquine-resistance gene pfcrt. Malar J. 2012;11:92. doi: 10.1186/1475-2875-11-92.
    1. Pickard AL, Wongsrichanalai C, Purfield A, Kamwendo D, Emery K, Zalewski C, et al. Resistance to antimalarials in Southeast Asia and genetic polymorphisms in pfmdr1. Antimicrob Agents Chemother. 2003;47:2418–2423. doi: 10.1128/AAC.47.8.2418-2423.2003.
    1. Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC. The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin. Mol Biochem Parasitol. 2000;108:13–23. doi: 10.1016/S0166-6851(00)00201-2.
    1. Sisowath C, Strömberg J, Mårtensson A, Msellem M, Obondo C, Björkman A, et al. In vivo selection of Plasmodium falciparum pfmdr1 86 N coding alleles by artemether-lumefantrine (Coartem) J Infect Dis. 2005;191:1014–1017. doi: 10.1086/427997.
    1. Peacekeeping UN. UN Mission’s Summary detailed by Country; 2017.

Source: PubMed

Sponsorzy i współpracownicy

Warunki medyczne

Interwencje lekowe

3
Subskrybuj