• View in gallery
    Figure 1.

    Study areas showing the prevalence of both mutant and wild-type alleles at each site. (A) Plasmodium falciparum chloroquine resistance transporter (pfcrt) K76T; (B) Plasmodium falciparum multidrug resistance transporter 1 (pfmdr1) N86Y.

  • View in gallery
    Figure 2.

    Proportion comparison of the prevalence of (A) Plasmodium falciparum chloroquine resistance transporter (pfcrt) 76T and (B) Plasmodium falciparum multidrug resistance transporter 1 (pfmdr1) 86Y in the present study with previous published studies 26,3538 from Bangladesh. Samples from non-CHT (Chittagong Hill Tracts) districts (Netrokona, n = 8 and Moulvibazar, n = 16) were not included in this analysis. *P < 0.05, **P < 0.01, ***P < 0.001; proportion comparison test.

  • View in gallery
    Figure 3.

    Proportion comparison of the prevalence of Plasmodium falciparum multidrug resistance transporter 1 (pfmdr1) N86, 86Y in the present study with previous published studies 26,3638 from Bangladesh. Samples from non-CHT (Chittagong Hill Tracts) districts (Moulvibazar, n = 2) were not included in this analysis. **P < 0.01; proportion comparison test.

  • 1.

    World Health Organization , 2019. World Malaria Report 2019. Geneva, Switzerland: WHO.

  • 2.

    Imwong M et al. 2020. Molecular epidemiology of resistance to antimalarial drugs in the Greater Mekong subregion: an observational study. Lancet Infect Dis (Epub ahead of print). Available at: https://doi.org/10.1016/S1473-3099(20)30228-0.

    • Search Google Scholar
    • Export Citation
  • 3.

    Bhumiratana A , Intarapuk A , Sorosjinda-Nunthawarasilp P , Maneekan P , Koyadun S , 2013. Border malaria associated with multidrug resistance on Thailand-Myanmar and Thailand-Cambodia borders: transmission dynamic, vulnerability, and surveillance. Biomed Res Int 2013: 363417.

    • Search Google Scholar
    • Export Citation
  • 4.

    Eyles DE , Hoo CC , Warren M , Sandosham AA , 1963. Plasmodium falciparum resistant to chloroquine in Cambodia. Am J Trop Med Hyg 12: 840843.

    • Search Google Scholar
    • Export Citation
  • 5.

    Thu AM , Phyo AP , Landier J , Parker DM , Nosten FH , 2017. Combating multidrug-resistant Plasmodium falciparum malaria. Febs J 284: 25692578.

    • Search Google Scholar
    • Export Citation
  • 6.

    World Health Organization , 2015. World Malaria Report 2015. Geneva, Switzerland: WHO.

  • 7.

    Noe A , Zaman SI , Rahman M , Saha AK , Aktaruzzaman MM , Maude RJ , 2018. Mapping the stability of malaria hotspots in Bangladesh from 2013 to 2016. Malar J 17: 259.

    • Search Google Scholar
    • Export Citation
  • 8.

    National Malaria Elimination Programme , 2019. National Malaria Data Bangladesh, 2007–2018. Dhaka, Bangladesh: Directorate General of Health Services.

    • Search Google Scholar
    • Export Citation
  • 9.

    Alam MS , Elahi R , Mohon AN , Al-Amin HM , Kibria MG , Khan WA , Khanum H , Haque R , 2016. Plasmodium falciparum genetic diversity in Bangladesh does not suggest a hypoendemic population structure. Am J Trop Med Hyg 94: 12451250.

    • Search Google Scholar
    • Export Citation
  • 10.

    Chang HH et al. 2019. Mapping imported malaria in Bangladesh using parasite genetic and human mobility data. Elife 8: e43481.

  • 11.

    World Health Organization , 2006. Guidelines for the Treatment of Malaria. Geneva, Switzerland: WHO.

  • 12.

    World Health Organization , 2019. Global Database on Antimalarial Drug Efficacy and Resistance. Geneva, Switzerland: WHO. Available at: https://www.who.int/malaria/areas/drug_resistance/drug_efficacy_database/en/. Accessed July 30, 2019.

    • Search Google Scholar
    • Export Citation
  • 13.

    DGHS , 2008. Strategic Plan for Malaria Control Programme Bangladesh. Dhaka, Bangladesh: Directorate General of Health Services.

  • 14.

    Haque U et al. 2014. Malaria burden and control in Bangladesh and prospects for elimination: an epidemiological and economic assessment. Lancet Glob Health 2: e98e105.

    • Search Google Scholar
    • Export Citation
  • 15.

    World Health Organization , 2018. Artemisinin Resistance and Artemisinin-Based Combination Therapy Efficacy. Geneva, Switzerland: WHO.

  • 16.

    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: 26192620.

    • Search Google Scholar
    • Export Citation
  • 17.

    Ariey F et al. 2014. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505: 5055.

  • 18.

    Phyo AP et al. 2012. Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. Lancet 379: 19601966.

    • Search Google Scholar
    • Export Citation
  • 19.

    Straimer J et al. 2015. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates. Science 347: 428431.

    • Search Google Scholar
    • Export Citation
  • 20.

    Menard D , Dondorp A , 2017. Antimalarial drug resistance: a threat to malaria elimination. Cold Spring Harb Perspect Med 7: a025619.

  • 21.

    Thriemer K , Starzengruber P , Khan WA , Haque R , Marma AS , Ley B , Vossen MG , Swoboda P , Akter J , Noedl H , 2010. Azithromycin combination therapy for the treatment of uncomplicated falciparum malaria in Bangladesh: an open-label randomized, controlled clinical trial. J Infect Dis 202: 392398.

    • Search Google Scholar
    • Export Citation
  • 22.

    Haque R et al. 2007. Therapeutic efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in Bangladesh. Am J Trop Med Hyg 76: 3941.

    • Search Google Scholar
    • Export Citation
  • 23.

    Samad R , Rahman MR , Yunus EB , Hussain MA , Arif SM , Islam MN , Hafiz SA , Hossain MM , Faiz MA , 2013. An open randomized controlled trial to compare the efficacy of two fixed dose combinations of artemesinin based combinations for uncomplicated falciparum malaria in Bangladesh. Bangladesh Med Res Counc Bull 39: 109115.

    • Search Google Scholar
    • Export Citation
  • 24.

    van den Broek IV et al. 2005. Efficacy of chloroquine + sulfadoxine--pyrimethamine, mefloquine + artesunate and artemether + lumefantrine combination therapies to treat Plasmodium falciparum malaria in the Chittagong Hill Tracts, Bangladesh. Trans R Soc Trop Med Hyg 99: 727735.

    • Search Google Scholar
    • Export Citation
  • 25.

    Rahman MM et al. 2008. Adherence and efficacy of supervised versus non-supervised treatment with artemether/lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in Bangladesh: a randomised controlled trial. Trans R Soc Trop Med Hyg 102: 861867.

    • Search Google Scholar
    • Export Citation
  • 26.

    Alam MS , Ley B , Nima MK , Johora FT , Hossain ME , Thriemer K , Auburn S , Marfurt J , Price RN , Khan WA , 2017. Molecular analysis demonstrates high prevalence of chloroquine resistance but no evidence of artemisinin resistance in Plasmodium falciparum in the Chittagong Hill Tracts of Bangladesh. Malar J 16: 335.

    • Search Google Scholar
    • Export Citation
  • 27.

    Directorate General of Health Services , 2018. Revised (Current) Malaria Treatment Regimen-2017. Dhaka, Bangladesh: Directorate General of Health Services.

    • Search Google Scholar
    • Export Citation
  • 28.

    Djimde A et al. 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344: 257263.

  • 29.

    Veiga MI , Dhingra SK , Henrich PP , Straimer J , Gnadig N , Uhlemann AC , Martin RE , Lehane AM , Fidock DA , 2016. Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies. Nat Commun 7: 11553.

    • Search Google Scholar
    • Export Citation
  • 30.

    Malmberg M , Ferreira PE , Tarning J , Ursing J , Ngasala B , Bjorkman A , Martensson A , Gil JP , 2013. Plasmodium falciparum drug resistance phenotype as assessed by patient antimalarial drug levels and its association with pfmdr1 polymorphisms. J Infect Dis 207: 842847.

    • Search Google Scholar
    • Export Citation
  • 31.

    Sidhu AB , Uhlemann AC , Valderramos SG , Valderramos JC , Krishna S , Fidock DA , 2006. Decreasing pfmdr1 copy number in Plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J Infect Dis 194: 528535.

    • Search Google Scholar
    • Export Citation
  • 32.

    Price RN et al. 2006. Molecular and pharmacological determinants of the therapeutic response to artemether-lumefantrine in multidrug-resistant Plasmodium falciparum malaria. Clin Infect Dis 42: 15701577.

    • Search Google Scholar
    • Export Citation
  • 33.

    Asua V , Vinden J , Conrad MD , Legac J , Kigozi SP , Kamya MR , Dorsey G , Nsobya SL , Rosenthal PJ , 2019. Changing molecular markers of antimalarial drug sensitivity across Uganda. Antimicrob Agents Chemother 63: e01818-18.

    • Search Google Scholar
    • Export Citation
  • 34.

    Mohon AN , Alam MS , Bayih AG , Folefoc A , Shahinas D , Haque R , Pillai DR , 2014. Mutations in Plasmodium falciparum K13 propeller gene from Bangladesh (2009–2013). Malar J 13: 431.

    • Search Google Scholar
    • Export Citation
  • 35.

    Kawai A , Arita N , Matsumoto Y , Kawabata M , Chowdhury MS , Saito-Ito A , 2011. Efficacy of chloroquine plus primaquine treatment and pfcrt mutation in uncomplicated falciparum malaria patients in Rangamati, Bangladesh. Parasitol Int 60: 341346.

    • Search Google Scholar
    • Export Citation
  • 36.

    Marma AS , Mita T , Eto H , Tsukahara T , Sarker S , Endo H , 2010. High prevalence of sulfadoxine/pyrimethamine resistance alleles in Plasmodium falciparum parasites from Bangladesh. Parasitol Int 59: 178182.

    • Search Google Scholar
    • Export Citation
  • 37.

    Srimuang K , Miotto O , Lim P , Fairhurst RM , Kwiatkowski DP , Woodrow CJ , Imwong M , 2016. Analysis of anti-malarial resistance markers in pfmdr1 and pfcrt across Southeast Asia in the tracking resistance to artemisinin collaboration. Malar J 15: 541.

    • Search Google Scholar
    • Export Citation
  • 38.

    van den Broek IV , van der Wardt S , Talukder L , Chakma S , Brockman A , Nair S , Anderson TC , 2004. Drug resistance in Plasmodium falciparum from the Chittagong Hill Tracts, Bangladesh. Trop Med Int Health 9: 680687.

    • Search Google Scholar
    • Export Citation
  • 39.

    Akter J , Thriemer K , Khan WA , Sullivan DJ Jr. , Noedl H , Haque R , 2012. Genotyping of Plasmodium falciparum using antigenic polymorphic markers and to study anti-malarial drug resistance markers in malaria endemic areas of Bangladesh. Malar J 11: 386.

    • Search Google Scholar
    • Export Citation
  • 40.

    Snounou G , Singh B , 2002. Nested PCR analysis of Plasmodium parasites. Methods Mol Med 72: 189203.

  • 41.

    Alam MS , Mohon AN , Mustafa S , Khan WA , Islam N , Karim MJ , Khanum H , Sullivan DJ Jr. , Haque R , 2011. Real-time PCR assay and rapid diagnostic tests for the diagnosis of clinically suspected malaria patients in Bangladesh. Malar J 10: 175.

    • Search Google Scholar
    • Export Citation
  • 42.

    Veiga MI , Ferreira PE , Bjorkman A , Gil JP , 2006. Multiplex PCR-RFLP methods for pfcrt, pfmdr1 and pfdhfr mutations in Plasmodium falciparum. Mol Cell Probes 20: 100104.

    • Search Google Scholar
    • Export Citation
  • 43.

    Duraisingh MT , Jones P , Sambou I , von Seidlein L , Pinder M , Warhurst DC , 2000. 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 108: 1323.

    • Search Google Scholar
    • Export Citation
  • 44.

    Ferreira ID , Rosario VE , Cravo PV , 2006. Real-time quantitative PCR with SYBR Green I detection for estimating copy numbers of nine drug resistance candidate genes in Plasmodium falciparum. Malar J 5: 1.

    • Search Google Scholar
    • Export Citation
  • 45.

    WWARN , 2014. Copy Number Estimation of the Plasmodium falciparum Pfmdr1 Gene. Available at: https://www.wwarn.org/tools-resources/procedures. Accessed July 2, 2018.

    • Search Google Scholar
    • Export Citation
  • 46.

    Laufer MK , Thesing PC , Eddington ND , Masonga R , Dzinjalamala FK , Takala SL , Taylor TE , Plowe CV , 2006. Return of chloroquine antimalarial efficacy in Malawi. N Engl J Med 355: 19591966.

    • Search Google Scholar
    • Export Citation
  • 47.

    Mwanza S , Joshi S , Nambozi M , Chileshe J , Malunga P , Kabuya JB , Hachizovu S , Manyando C , Mulenga M , Laufer M , 2016. The return of chloroquine-susceptible Plasmodium falciparum malaria in Zambia. 15: 584.

    • Search Google Scholar
    • Export Citation
  • 48.

    Mohammed A et al. 2013. Trends in chloroquine resistance marker, Pfcrt-K76T mutation ten years after chloroquine withdrawal in Tanzania. Malar J 12: 415.

    • Search Google Scholar
    • Export Citation
  • 49.

    Huang B et al. 2016. Prevalence of crt and mdr-1 mutations in Plasmodium falciparum isolates from Grande Comore island after withdrawal of chloroquine. Malar J 15: 414.

    • Search Google Scholar
    • Export Citation
  • 50.

    Gharbi M et al. 2013. Longitudinal study assessing the return of chloroquine susceptibility of Plasmodium falciparum in isolates from travellers returning from West and Central Africa, 2000-2011. Malar J 12: 35.

    • Search Google Scholar
    • Export Citation
  • 51.

    Awasthi G , Prasad GB , Das A , 2011. Population genetic analyses of Plasmodium falciparum chloroquine receptor transporter gene haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 41: 705709.

    • Search Google Scholar
    • Export Citation
  • 52.

    Venkatesan M et al. 2014. Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine. Am J Trop Med Hyg 91: 833843.

    • Search Google Scholar
    • Export Citation
  • 53.

    Sisowath C , Strömberg J , Mårtensson A , Msellem M , Obondo C , Björkman A , Gil JP , 2005. In vivo selection of Plasmodium falciparum pfmdr1 86N coding alleles by artemether-lumefantrine (Coartem). J Infect Dis 191: 10141017.

    • Search Google Scholar
    • Export Citation
  • 54.

    Ljolje D et al. 2018. Prevalence of molecular markers of artemisinin and lumefantrine resistance among patients with uncomplicated Plasmodium falciparum malaria in three provinces in Angola, 2015. Malar J 17: 84.

    • Search Google Scholar
    • Export Citation
  • 55.

    Humphreys GS , Merinopoulos I , Ahmed J , Whitty CJ , Mutabingwa TK , Sutherland CJ , Hallett RL , 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: 991997.

    • Search Google Scholar
    • Export Citation
  • 56.

    Baliraine FN , Rosenthal PJ , 2011. Prolonged selection of pfmdr1 polymorphisms after treatment of falciparum malaria with artemether-lumefantrine in Uganda. J Infect Dis 204: 11201124.

    • Search Google Scholar
    • Export Citation
  • 57.

    Happi CT , Gbotosho GO , Folarin OA , Sowunmi A , Hudson T , O'Neil M , Milhous W , Wirth DF , Oduola AM , 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: 888895.

    • Search Google Scholar
    • Export Citation
  • 58.

    Price RN et al. 2004. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet 364: 438447.

  • 59.

    Noedl H , Faiz MA , Yunus EB , Rahman MR , Hossain MA , Samad R , Miller RS , Pang LW , Wongsrichanalai C , 2003. Drug-resistant malaria in Bangladesh: an in vitro assessment. Am J Trop Med Hyg 68: 140142.

    • Search Google Scholar
    • Export Citation
  • 60.

    Ménard D et al. 2016. A worldwide map of Plasmodium falciparum K13-propeller polymorphisms. N Engl J Med 374: 24532464.

Past two years Past Year Past 30 Days
Abstract Views 447 0 0
Full Text Views 493 289 26
PDF Downloads 374 181 11
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

 

 

Persistence of Markers of Chloroquine Resistance in Plasmodium falciparum in Bangladesh

Fatema Tuj Johora Infectious Diseases Division, International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), Dhaka, Bangladesh;

Search for other papers by Fatema Tuj Johora in
Current site
Google Scholar
PubMed
Close
,
Rubayet Elahi Infectious Diseases Division, International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), Dhaka, Bangladesh;
Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland;

Search for other papers by Rubayet Elahi in
Current site
Google Scholar
PubMed
Close
,
Maisha Khair Nima Infectious Diseases Division, International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), Dhaka, Bangladesh;
Department of Biological Sciences, Boler-Parseghian Center for Rare and Neglected Diseases, University of Notre Dame, Notre Dame, Indiana;

Search for other papers by Maisha Khair Nima in
Current site
Google Scholar
PubMed
Close
,
Mohammad Sharif Hossain Infectious Diseases Division, International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), Dhaka, Bangladesh;

Search for other papers by Mohammad Sharif Hossain in
Current site
Google Scholar
PubMed
Close
,
Humaira Rashid Infectious Diseases Division, International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), Dhaka, Bangladesh;

Search for other papers by Humaira Rashid in
Current site
Google Scholar
PubMed
Close
,
Mohammad Golam Kibria Infectious Diseases Division, International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), Dhaka, Bangladesh;

Search for other papers by Mohammad Golam Kibria in
Current site
Google Scholar
PubMed
Close
,
Abu Naser Mohon Infectious Diseases Division, International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), Dhaka, Bangladesh;
Department of Microbiology, Immunology and Infectious Disease, University of Calgary, Alberta, Canada

Search for other papers by Abu Naser Mohon in
Current site
Google Scholar
PubMed
Close
,
Wasif A. Khan Infectious Diseases Division, International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), Dhaka, Bangladesh;

Search for other papers by Wasif A. Khan in
Current site
Google Scholar
PubMed
Close
,
Rashidul Haque Infectious Diseases Division, International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), Dhaka, Bangladesh;

Search for other papers by Rashidul Haque in
Current site
Google Scholar
PubMed
Close
, and
Mohammad Shafiul Alam Infectious Diseases Division, International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), Dhaka, Bangladesh;

Search for other papers by Mohammad Shafiul Alam in
Current site
Google Scholar
PubMed
Close

ABSTRACT

The control of malaria, in terms of drug resistance, remains a significant global challenge, with Bangladesh, a malaria-endemic country, being no exception. The aim of this study was to explore antimalarial resistance in Bangladesh by molecular analysis of Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance transporter 1 (pfmdr1) genetic markers of P. falciparum. Samples were obtained from uncomplicated malaria patients between 2009 and 2014 from six malaria-endemic districts. Based on parasite transmission intensity, the endemic districts were divided into high-transmission (Chittagong Hill Tracts [CHT]) and low-transmission (non-CHT) regions. Falciparum malaria-positive isolates were genotyped for K76T of the pfcrt gene, and N86Y and Y184F of the pfmdr1 gene: in total, 262 P. falciparum clinical isolates were analyzed. In CHT areas, the prevalence of polymorphisms was 70.6% for 76T, 14.4% for 86Y, and 7.8% for 184F. In non-CHT areas, 76T and 86Y mutations were found in 78.0% and 19.5% of the samples, respectively, whereas no 184F mutations were observed. We compared our data with previous similar molecular observations, which shows a significant decrease in pfcrt 76T mutation prevalence. No pfmdr1 amplification was observed in any of the samples suggesting an unaltered susceptibility to amino alcohol drugs such as mefloquine and lumefantrine. This study provides an updated assessment of the current status of pfcrt and pfmdr1 gene mutations in Bangladesh, and suggests there is persistent high prevalence of markers of resistance to aminoquinoline drugs.

INTRODUCTION

In 2018, Plasmodium falciparum contributed to 50% of all reported malaria cases in Southeast Asia (SEA). 1 Southeast Asia has always been an important area for the observation of antimalarial drug resistance, as this region contains the hotspot zone (Greater Mekong Sub-region) 2 for the onset and spread of drug resistance against chloroquine ([CQ] a 4-aminoquinoline antimalarial) monotherapy, as well as to different multidrug combination therapies used in malaria treatment. 3,4 Plasmodium falciparum has demonstrated resistance to nearly every antimalarial drug, although in most regions, artemisinin (ART)-based combination therapies (ACTs) offer effective therapy 5 : in the absence of a vaccine, this resistance threatens the WHO Roll Back Malaria action plan in areas of high transmission. 6

Bangladesh, a malaria-endemic country in SEA, shares international borders with other malaria-endemic countries, such as India and Myanmar. In 2016, it had an annual parasitic index of 2.08/1,000 population from 13 endemic districts. 7 According to the national malaria elimination program (NMEP), there were a total of 107,226 malaria cases between 2015 and 2018 in Bangladesh, 88% of which were associated with P. falciparum alone, or in combination with Plasmodium vivax. 8 More than 90% of these cases occurred in the high-transmission areas of Bandarban, Khagrachhari, and Rangamati (regionally known as Chittagong Hill Tracts [CHT]), 810 and other cases have been reported from non-CHT (low-transmission) 8 areas.

In malaria-endemic countries, the WHO recommends ACTs as the frontline treatment for uncomplicated malaria. 11 The ACT, artemether–lumefantrine (AL), has excellent efficacy in the SEA region 12 and has been recommended as the firstline treatment in Bangladesh since 2004. 13,14 The fast-acting component of ACTs, ART, or its derivatives dihydroartemisinin (DHA), artesunate, and artemether, works in combination with various partner drugs, such as amino alcohols (lumefantrine [LUM] and mefloquine [MQ]), 4-aminoquinolines (amodiaquine, piperaquine, and pyronaridine), and a sulfa-antifolate combination (sulfadoxine–pyrimethamine). 15 The first documentation of ART resistance was reported in western Cambodia 16 ; this was followed by the discovery of multiple kelch13 gene mutations, 17 which eventually spread as well as emerged independently to the rest of mainland SEA. 18 Failure of ART treatment is defined by delayed parasite clearance. 19 ART treatment failure also selects for ACT partner drug resistance. 20 In Bangladesh, clinical trials of ACTs, for example, AL, artesunate–mefloquine, artesunate–amodiaquine, and artesunate–azithromycin, have shown excellent efficacy, with rapid parasite clearance and high rates of treatment success. 2125 Although AL is the mandated treatment for uncomplicated malaria, CQ remains available as an over-the-counter medication for self-treatment of malaria, as well as for vivax malaria treatment in some parts of the country. 26,27

Several drug resistance mechanisms have been reported in P. falciparum; CQ resistance (CQR) has been linked to the mutation in the P. falciparum chloroquine resistance transporter gene (pfcrt) at codon 76. 28 A single nucleotide polymorphism in the P. falciparum multidrug resistance transporter 1 gene (pfmdr1) at codon 86 is associated with hypersensitivity to LUM, MQ, and also to DHA, an active artemisinin metabolite. 29 Mutation at codon 86 is also associated with decreased susceptibility to aminoquinolines. 29 In addition, some studies also reported the association of mutation at codon 184 with parasite susceptibility to various antimalarials. 29,30 A copy number increase in this gene is also associated with decreased susceptibility to LUM. 3133

Recent studies found no kelch13 gene mutations in P. falciparum from Bangladesh. 34,26 A handful of studies have also investigated CQR-associated mutations from specific endemic districts in Bangladesh. 26,3539 However, there has not been a large-scale study of the currently used antimalarials, CQ and AL, resistance-associated molecular markers, in Bangladesh. Hence, in this study, we examined the prevalence of pfcrt and pfmdr1 markers for resistance to various antimalarials in clinical samples across the malaria-endemic districts of Bangladesh.

METHODS

Study sites.

Samples were collected from 11 subdistricts (Upazilas) in three CHT areas: Bandarban (subdistricts: Bandarban Sadar, Naikhongchhari, and Lama), Khagrachhari (subdistrict: Matiranga), and Rangamati (subdistrict: Rajasthali); and three non-CHT areas: Cox’s Bazar (subdistricts: Ramu and Ukhiya), Moulvibazar (subdistricts: Kamalganj and Sreemangal), and Netrokona (subdistricts: Kalmakanda and Durgapur) (Figure 1). All the sites share an international border with malaria-endemic countries, either India or Myanmar.

Figure 1.
Figure 1.

Study areas showing the prevalence of both mutant and wild-type alleles at each site. (A) Plasmodium falciparum chloroquine resistance transporter (pfcrt) K76T; (B) Plasmodium falciparum multidrug resistance transporter 1 (pfmdr1) N86Y.

Citation: The American Journal of Tropical Medicine and Hygiene 104, 1; 10.4269/ajtmh.20-0415

Sample collection.

Blood samples were collected directly into an EDTA tube, from adults (5 mL) and children (3 mL) with clinical symptoms of malaria, and stored at −20°C. A total of 296 microscopy-positive falciparum malaria samples were collected. DNA extraction was performed using the QIAamp Blood Mini Kit (Qiagen Inc., Hilden, Germany) following the manufacturer’s instructions for collected whole blood. To determine mono-infection of P. falciparum, PCR 40 was performed for all the samples, along with real-time quantitative PCR (qPCR) confirmation, following the methods in Alam et al. 41 A total of 262 samples (CHT: 180; non-CHT: 82) were confirmed as P. falciparum mono-infection. These samples were selected for drug resistance marker analysis. The institutional Ethics Review Committee of the International Centre for Diarrhoeal Disease Research, Bangladesh, approved the original study. All study participants provided their informed consent for future use of the sample.

Genotyping of pfcrt and pfmdr1.

All DNA samples were analyzed for pfcrt and pfmdr1 gene mutations using PCR and restriction fragment length polymorphism. Polymerase, and all the restriction enzymes were obtained from New England Biolabs Inc. (Ipswich, MA). Analysis of the K76T mutation in the pfcrt gene was conducted by the method described by Veiga et al. 42 In brief, the pfcrt gene containing codon 76 was amplified by nested PCR, resulting in a 145-bp amplicon. This amplicon was then restriction digested with the ApoI enzyme. In the absence of 76T mutation (145 bp mutant allele), the PCR amplicon results in 98 bp and 47 bp fragments.

To analyze mutations of codons 86 and 184 in the pfmdr1 gene, the protocol described by Duraisingh et al. 43 was followed. In brief, the pfmdr1 gene fragment containing codons 86 and 184 was amplified by nested PCR, resulting in a 560-bp amplicon. This amplicon was subsequently digested with restriction enzymes, ApoI and DraI. In the absence of the 86Y mutant allele, the ApoI restriction digestion generates 249 bp and 256 bp fragments, whereas DraI digestion in the presence of the 184F mutant allele results in 242 bp, 173 bp, and 114 bp fragments. For both pfcrt and pfmdr1, a sample can show heterozygosity, that is, can contain both wild-type and mutant alleles, which suggests infection by at least two genotypically different parasites. All the PCR and restriction digestion products were resolved on 2–2.5% agarose gels containing 0.1 μg/mL ethidium bromide (Invitrogen Life Technologies, Carlsbad, CA).

Plasmodium falciparum multidrug resistance transporter 1 copy number analysis.

The pfmdr1 copy number was estimated using qPCR, following the method described by Ferreira et al. 44 In brief, in a final 10 μL PCR reaction volume, 5 μL iQ™ SYBR® Green Supermix (BioRad Laboratories Inc., Hercules, CA), 300 nM of each forward and reverse oligonucleotide (primer) (Integrated DNA Technologies Inc., Coralville, IA), and 2 μL of template DNA were added. In each run of qPCR, DNA from P. falciparum clone 3D7 (which has a copy number of 1) was used as a calibrator, and P. falciparum clone Dd2 DNA (which has a copy number of ∼4) was used as an amplification control for multiple copies of pfmdr1. The P. falciparum clones 3D7 and Dd2 DNA were from MR4 (BEI resources, Manassas, VA). We used the P. falciparum β-actin gene as the reference gene for this experiment. All samples were run in duplicate. The ΔΔCt method was used for the analysis of qPCR results, where ΔΔCt = (Ct of pfmdr1 gene - Ct of reference gene) of the sample - (Ct of pfmdr1 gene - Ct of reference gene) of the 3D7 calibrator. The copy number of pfmdr1 was calculated as 2−ΔΔCt; a copy number of 1.5 was set as a threshold to define multiple copies of pfmdr1. 45

Statistical analysis.

The prevalence of pfcrt and pfmdr1 alleles within CHT and non-CHT areas was computed in Microsoft Excel (Microsoft Corporation, Redmond, WA). A proportion comparison test was performed using Stata (version 15.1, Stata Corp., College Station, TX) to observe any changes between the resistance status of these areas. The statistical significance level was defined as P < 0.05.

RESULTS

Plasmodium falciparum chloroquine resistance transporter and pfmdr1 polymorphisms.

We identified pfcrt 76T mutations in 191 of 262 samples (72.9%), from all the study areas (Table 1). From the 180 CHT samples, the 76T mutation was observed in 127 samples (70.6%), whereas 64 of the 82 non-CHT samples (78.0%) had the 76T mutation. No mixed alleles were recorded in any of the isolates for the pfcrt 76 position. The proportion comparison test showed there was no significant difference (P > 0.05) in the prevalence of the pfcrt 76T mutation between CHT and non-CHT regions.

Table 1

Prevalence of polymorphisms in pfcrt and pfmdr1 genes.

pfcrt, n (%) pfmdr1, n (%)
Polymorphism 76T 86Y 184F N86, 86Y
Total (n = 262) 191 (72.9) 42 (16.0) 14 (5.3) 40 (15.3)
CHT region (n = 180) 127 (70.6) 26 (14.4) 14 (7.8) 31 (17.2)
 Bandarban (n = 24) 21 (87.5) 4 (16.7) 0 (0.0) 2 (8.3)
 Khagrachhari (n = 151) 102 (67.5) 21 (13.9) 14 (9.3) 28 (18.5)
 Rangamati (n = 5) 4 (80.0) 1 (20.0) 0 (0.0) 1 (20.0)
Non-CHT region (n = 82) 64 (78.0) 16 (19.5) 0 (0.0) 9 (11.0)
 Cox’s Bazar (n = 58) 44 (75.9) 8 (13.8) 0 (0.0) 7 (12.1)
 Moulvibazar (n = 16) 13 (81.3) 5 (31.3) 0 (0.0) 2 (12.5)
 Netrokona (n = 8) 7 (87.5) 3 (37.5) 0 (0.0) 0 (0.0)

CHT = Chittagong Hill Tracts; pfcrt = Plasmodium falciparum chloroquine resistance transporter; pfmdr1 = Plasmodium falciparum multidrug resistance transporter 1.

For pfmdr1, we evaluated polymorphisms in the N86 and Y184 positions in all the samples (262) and found the 86Y mutant allele in 42 (16.0%) and the 184F mutant allele in 14 (5.3%) (Table 1). Most of the 86Y mutations and all the 184F mutations were from the CHT area (Khagrachhari district). We also found N86 and 86Y mixed alleles in 40 samples (15.3%), which suggests a mixed infection in these samples. As for the mixed allele, 31 (17.2%) of the mixed alleles were from the CHT region. A proportion comparison of both 86Y and mixed mutations between the CHT and non-CHT regions did not show any significant difference (P > 0.05) in the prevalence of these mutations between the regions.

For the analysis of the pfmdr1 haplotype for polymorphic positions 86 and 184 (Table 2), we did not include the samples with the mixed allele for position 86. We found N86Y184 was the most prevalent in both the CHT (82.6%) and non-CHT (78.1%) regions, whereas Y86Y184 was more prevalent (22.0%) in the non-CHT region. Y86F184 haplotypes were only found in CHT regions (3.4%).

Table 2

Plasmodium falciparum multidrug resistance transporter 1 haplotypes for position 86 and 184.

pfmdr1 haplotype Total (n = 262) CHT region (n = 149) Non-CHT region (n = 73)
86 184 Frequency of isolates Frequency of isolates Frequency of isolates
N Y 180 123 (82.6%) 57 (78.1%)
N F ND ND ND
Y Y 37 21 (14.1%) 16 (22.0%)
Y F 5 5 (3.4%) ND

CHT = Chittagong Hill Tracts; ND = not detected; pfmdr1 = Plasmodium falciparum multidrug resistance transporter 1. Samples with mixed infection at position 86 (CHT: 31; non-CHT: 9) were excluded for this analysis.

Changes in resistance markers over time.

Over the last two decades, several molecular studies have been conducted using pfcrt K76T and pfmdr1 N86Y markers to investigate malaria drug resistance in the malaria-endemic districts of Rangamati, 35 Bandarban, 26,36 Khagrachhari, 38 and Cox’s Bazar. 37 We used a proportion comparison test to compare our data with these published studies to observe the changes, if any, in the prevalence of antimalarial resistance–associated molecular markers (Figure 2). This analysis revealed a persistently high prevalence of the pfcrt 76T allele and a low prevalence of the pfmdr1 86Y allele. We observed a decreasing trend in pfcrt 76T mutation in Khagrachhari, Bandarban, and Cox’s Bazar districts over the last two decades (Figure 2A). Likewise, the pfmdr1 86Y mutation in the Khagrachhari district declined (Figure 2B). As for pfmdr1 184F mutations, there were no previous studies in Khagrachhari; therefore, we could not do a proportion comparison test for this mutation.

Figure 2.
Figure 2.

Proportion comparison of the prevalence of (A) Plasmodium falciparum chloroquine resistance transporter (pfcrt) 76T and (B) Plasmodium falciparum multidrug resistance transporter 1 (pfmdr1) 86Y in the present study with previous published studies 26,3538 from Bangladesh. Samples from non-CHT (Chittagong Hill Tracts) districts (Netrokona, n = 8 and Moulvibazar, n = 16) were not included in this analysis. *P < 0.05, **P < 0.01, ***P < 0.001; proportion comparison test.

Citation: The American Journal of Tropical Medicine and Hygiene 104, 1; 10.4269/ajtmh.20-0415

Mixed alleles of polymorphic pfmdr1 N86 have been reported previously, 26,3538 although these studies have either excluded or apportioned the mixed population of pfmdr1 N86, 86Y for their analysis. In this study, we compared only the mixed pfmdr1 N86, 86Y population with mixed populations from previous studies, using a proportion comparison test for each district (Figure 3). We found that the prevalence of mixed alleles in the Khagrachhari district has significantly increased.

Figure 3.
Figure 3.

Proportion comparison of the prevalence of Plasmodium falciparum multidrug resistance transporter 1 (pfmdr1) N86, 86Y in the present study with previous published studies 26,3638 from Bangladesh. Samples from non-CHT (Chittagong Hill Tracts) districts (Moulvibazar, n = 2) were not included in this analysis. **P < 0.01; proportion comparison test.

Citation: The American Journal of Tropical Medicine and Hygiene 104, 1; 10.4269/ajtmh.20-0415

Plasmodium falciparum multidrug resistance transporter 1 copy number.

Amplification of the pfmdr1 gene to investigate copy number increase was successful in 256 of the 262 isolates (97.7% [CHT: 176; non-CHT: 80]). The control DNA gave reproducible results, as 3D7 and Dd2 had a mean copy number of 1.0 and 3.0, respectively. 45 Using a copy number threshold of 1.5 to define multiple copies, 45 when rounded to the nearest integer, all the isolates were found to contain a single gene copy with a mean of 0.8 (SD = ±0.3).

DISCUSSION

In 2004, the NMEP introduced AL as an alternative to CQ, to treat uncomplicated falciparum malaria in Bangladesh; however, it took 3 years to implement AL treatment in all 13 malaria-endemic districts. 14 This study and previous studies demonstrate that the CQR allele is dominant in Bangladesh (Figure 2A). Among the study districts, Khagrachhari, Bandarban, and Cox’s Bazar showed a significant decrease in CQR over time (Figure 2A). Some African countries have shown a reversion to CQ sensitivity in P. falciparum isolates after the complete cessation of CQ use. 4650 A limitation of this study is that complete pfcrt haplotype data are not available in Bangladesh; therefore, the possibility of a reversion to CQ sensitivity cannot be addressed. Cox’s Bazar (on the Myanmar border) has more than 95% CVIET haplotype frequency, which is similar to Myanmar, 37 whereas in India (which borders other study districts), SVMNK prevails over CVIET, in terms of haplotype frequency. 51 Thus, the different endemic districts of Bangladesh, which are surrounded by endemic countries, may display different pfcrt haplotypes. However, despite the fact that CQ has not been used for falciparum malaria treatment for more than a decade, it is still used for the treatment of mono-infection of P. vivax. 27 As a result, CQ remains accessible via local drug store, reducing the likelihood of the CQR allele reverting to the sensitive allele in Bangladesh.

In this study, we found that the pfmdr1 86Y mutation occurred in 14.4% of samples from the CHT region (Table 1); another recent study in a CHT subdistrict found 13.9% occurrence. 26 The presence of the 86Y mutant allele can be attributed to the circulating CQ pressure; this mutation leads to decreased susceptibility to CQ. 29 However, there was a significant decrease in pfmdr1 86Y prevalence in the Khagrachhari district (Figure 2B), which could potentially facilitate the reversion of CQ sensitivity in this district.

The pfmdr1 86Y mutation has been linked to hypersensitivity to LUM, MQ, and DHA. 29,32,33 The stable prevalence of pfmdr1 86Y mutation over the last two decades in Bangladesh suggests susceptibility to LUM (the partner drug for ACT in Bangladesh) is unaffected. However, the significant increase in the prevalence of the wild-type pfmdr1 N86 allele in Khagrachhari (Figure 2B) is of significant concern, as it has been reported that parasites with the N86 allele have reduced susceptibility to LUM, compared with parasites with the 86Y allele. 52,53 The pfmdr1 N86 mutation cannot be used as a predictive marker for LUM resistance on its own 54 ; several studies have reported the presence of pfmdr1 184F and D1246 markers, along with N86 after prolonged AL treatment. 30,5557 In our study, we observed a low prevalence of the pfmdr1 184F mutation, which is consistent with other studies. 37,39 This mutation appears to have a weaker relationship with drug susceptibility 29,30,58 and may not by itself cause the failure of drug treatment. However, a clinical tolerance is observed to aminoquinolines when 184F is present with N86 mutation in parasites with predominant Asian/African variant of pfcrt. 29 We did not analyze our sample for the pfmdr1 D1246Y polymorphism; future deep sequencing or targeted sequencing analyses might help identify new or known polymorphisms in the pfmdr1 gene of the circulating parasite population of Bangladesh.

In parasites from SEA, the circulating pfmdr1 haplotype was either N86F184 or N86Y184. 29 In this study, the N86Y184 haplotype has been observed at a high frequency in both the CHT and non-CHT regions (Table 2), which is similar to the findings from a previous study in the Cox’s Bazar and Bandarban regions. 39

A copy number increase of pfmdr1 is linked to resistance to LUM 32 and MQ. 3133 Although MQ is not the ACT partner drug in Bangladesh, Noedl et al. 59 showed a high prevalence of MQ resistance in Bangladesh, in vitro. They concluded this might be due to circulating parasite populations of inherent MQ resistance, or resistant parasite strains, from neighboring Myanmar. In our study, we did not observe an increase in the copy number of the pfmdr1 gene. This finding is consistent with a previous study in this region 37 and provides reassurance that the efficacy of LUM has not been affected.

In the past two decades, before and after the introduction of ACTs in Bangladesh, only a handful of molecular studies on drug-resistant markers of antimalarials have been conducted, focusing on either one or two endemic regions. In this study, we analyzed clinical samples from a wider geographical setting, covering both high- and low-transmission areas of the country. Regardless of the transmission area, there was no statistically significant difference between the prevalence of pfcrt and pfmdr1 markers of drug resistance.

A previous study from Bangladesh that investigated molecular markers for ART resistance, with this same sample set, did not report the presence of any established resistance markers, except for a kelch13 A578S mutation, which appears to not mediate resistance, in two isolates. 34,60 The monitoring of ART-resistant markers is important as AL is the frontline antimalarial treatment. It is also important to monitor partner drug (LUM) efficacy of this combination therapy in this region. Therefore, the real-time monitoring of pfmdr1 and pfcrt markers enables tracking the emergence of drug resistance in advance and allows the regional malaria control programs to make informed decisions to prevent rise of widespread treatment failure and/or resistance.

ACKNOWLEDGMENTS

The icddr,b acknowledges, with gratitude, the commitment of the Swedish International Development Cooperation Agency (SIDA), the Swiss Academy of Medical Sciences (SAMS), and Velux Stiftung to its research efforts. The icddr,b is also grateful to the governments of Bangladesh, Canada, Sweden, and the United Kingdom for providing core/unrestricted support. We would like to thank the handling editor and the reviewers for their insightful comments that helped us improve the manuscript.

REFERENCES

  • 1.

    World Health Organization , 2019. World Malaria Report 2019. Geneva, Switzerland: WHO.

  • 2.

    Imwong M et al. 2020. Molecular epidemiology of resistance to antimalarial drugs in the Greater Mekong subregion: an observational study. Lancet Infect Dis (Epub ahead of print). Available at: https://doi.org/10.1016/S1473-3099(20)30228-0.

    • Search Google Scholar
    • Export Citation
  • 3.

    Bhumiratana A , Intarapuk A , Sorosjinda-Nunthawarasilp P , Maneekan P , Koyadun S , 2013. Border malaria associated with multidrug resistance on Thailand-Myanmar and Thailand-Cambodia borders: transmission dynamic, vulnerability, and surveillance. Biomed Res Int 2013: 363417.

    • Search Google Scholar
    • Export Citation
  • 4.

    Eyles DE , Hoo CC , Warren M , Sandosham AA , 1963. Plasmodium falciparum resistant to chloroquine in Cambodia. Am J Trop Med Hyg 12: 840843.

    • Search Google Scholar
    • Export Citation
  • 5.

    Thu AM , Phyo AP , Landier J , Parker DM , Nosten FH , 2017. Combating multidrug-resistant Plasmodium falciparum malaria. Febs J 284: 25692578.

    • Search Google Scholar
    • Export Citation
  • 6.

    World Health Organization , 2015. World Malaria Report 2015. Geneva, Switzerland: WHO.

  • 7.

    Noe A , Zaman SI , Rahman M , Saha AK , Aktaruzzaman MM , Maude RJ , 2018. Mapping the stability of malaria hotspots in Bangladesh from 2013 to 2016. Malar J 17: 259.

    • Search Google Scholar
    • Export Citation
  • 8.

    National Malaria Elimination Programme , 2019. National Malaria Data Bangladesh, 2007–2018. Dhaka, Bangladesh: Directorate General of Health Services.

    • Search Google Scholar
    • Export Citation
  • 9.

    Alam MS , Elahi R , Mohon AN , Al-Amin HM , Kibria MG , Khan WA , Khanum H , Haque R , 2016. Plasmodium falciparum genetic diversity in Bangladesh does not suggest a hypoendemic population structure. Am J Trop Med Hyg 94: 12451250.

    • Search Google Scholar
    • Export Citation
  • 10.

    Chang HH et al. 2019. Mapping imported malaria in Bangladesh using parasite genetic and human mobility data. Elife 8: e43481.

  • 11.

    World Health Organization , 2006. Guidelines for the Treatment of Malaria. Geneva, Switzerland: WHO.

  • 12.

    World Health Organization , 2019. Global Database on Antimalarial Drug Efficacy and Resistance. Geneva, Switzerland: WHO. Available at: https://www.who.int/malaria/areas/drug_resistance/drug_efficacy_database/en/. Accessed July 30, 2019.

    • Search Google Scholar
    • Export Citation
  • 13.

    DGHS , 2008. Strategic Plan for Malaria Control Programme Bangladesh. Dhaka, Bangladesh: Directorate General of Health Services.

  • 14.

    Haque U et al. 2014. Malaria burden and control in Bangladesh and prospects for elimination: an epidemiological and economic assessment. Lancet Glob Health 2: e98e105.

    • Search Google Scholar
    • Export Citation
  • 15.

    World Health Organization , 2018. Artemisinin Resistance and Artemisinin-Based Combination Therapy Efficacy. Geneva, Switzerland: WHO.

  • 16.

    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: 26192620.

    • Search Google Scholar
    • Export Citation
  • 17.

    Ariey F et al. 2014. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505: 5055.

  • 18.

    Phyo AP et al. 2012. Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. Lancet 379: 19601966.

    • Search Google Scholar
    • Export Citation
  • 19.

    Straimer J et al. 2015. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates. Science 347: 428431.

    • Search Google Scholar
    • Export Citation
  • 20.

    Menard D , Dondorp A , 2017. Antimalarial drug resistance: a threat to malaria elimination. Cold Spring Harb Perspect Med 7: a025619.

  • 21.

    Thriemer K , Starzengruber P , Khan WA , Haque R , Marma AS , Ley B , Vossen MG , Swoboda P , Akter J , Noedl H , 2010. Azithromycin combination therapy for the treatment of uncomplicated falciparum malaria in Bangladesh: an open-label randomized, controlled clinical trial. J Infect Dis 202: 392398.

    • Search Google Scholar
    • Export Citation
  • 22.

    Haque R et al. 2007. Therapeutic efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in Bangladesh. Am J Trop Med Hyg 76: 3941.

    • Search Google Scholar
    • Export Citation
  • 23.

    Samad R , Rahman MR , Yunus EB , Hussain MA , Arif SM , Islam MN , Hafiz SA , Hossain MM , Faiz MA , 2013. An open randomized controlled trial to compare the efficacy of two fixed dose combinations of artemesinin based combinations for uncomplicated falciparum malaria in Bangladesh. Bangladesh Med Res Counc Bull 39: 109115.

    • Search Google Scholar
    • Export Citation
  • 24.

    van den Broek IV et al. 2005. Efficacy of chloroquine + sulfadoxine--pyrimethamine, mefloquine + artesunate and artemether + lumefantrine combination therapies to treat Plasmodium falciparum malaria in the Chittagong Hill Tracts, Bangladesh. Trans R Soc Trop Med Hyg 99: 727735.

    • Search Google Scholar
    • Export Citation
  • 25.

    Rahman MM et al. 2008. Adherence and efficacy of supervised versus non-supervised treatment with artemether/lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in Bangladesh: a randomised controlled trial. Trans R Soc Trop Med Hyg 102: 861867.

    • Search Google Scholar
    • Export Citation
  • 26.

    Alam MS , Ley B , Nima MK , Johora FT , Hossain ME , Thriemer K , Auburn S , Marfurt J , Price RN , Khan WA , 2017. Molecular analysis demonstrates high prevalence of chloroquine resistance but no evidence of artemisinin resistance in Plasmodium falciparum in the Chittagong Hill Tracts of Bangladesh. Malar J 16: 335.

    • Search Google Scholar
    • Export Citation
  • 27.

    Directorate General of Health Services , 2018. Revised (Current) Malaria Treatment Regimen-2017. Dhaka, Bangladesh: Directorate General of Health Services.

    • Search Google Scholar
    • Export Citation
  • 28.

    Djimde A et al. 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344: 257263.

  • 29.

    Veiga MI , Dhingra SK , Henrich PP , Straimer J , Gnadig N , Uhlemann AC , Martin RE , Lehane AM , Fidock DA , 2016. Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies. Nat Commun 7: 11553.

    • Search Google Scholar
    • Export Citation
  • 30.

    Malmberg M , Ferreira PE , Tarning J , Ursing J , Ngasala B , Bjorkman A , Martensson A , Gil JP , 2013. Plasmodium falciparum drug resistance phenotype as assessed by patient antimalarial drug levels and its association with pfmdr1 polymorphisms. J Infect Dis 207: 842847.

    • Search Google Scholar
    • Export Citation
  • 31.

    Sidhu AB , Uhlemann AC , Valderramos SG , Valderramos JC , Krishna S , Fidock DA , 2006. Decreasing pfmdr1 copy number in Plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J Infect Dis 194: 528535.

    • Search Google Scholar
    • Export Citation
  • 32.

    Price RN et al. 2006. Molecular and pharmacological determinants of the therapeutic response to artemether-lumefantrine in multidrug-resistant Plasmodium falciparum malaria. Clin Infect Dis 42: 15701577.

    • Search Google Scholar
    • Export Citation
  • 33.

    Asua V , Vinden J , Conrad MD , Legac J , Kigozi SP , Kamya MR , Dorsey G , Nsobya SL , Rosenthal PJ , 2019. Changing molecular markers of antimalarial drug sensitivity across Uganda. Antimicrob Agents Chemother 63: e01818-18.

    • Search Google Scholar
    • Export Citation
  • 34.

    Mohon AN , Alam MS , Bayih AG , Folefoc A , Shahinas D , Haque R , Pillai DR , 2014. Mutations in Plasmodium falciparum K13 propeller gene from Bangladesh (2009–2013). Malar J 13: 431.

    • Search Google Scholar
    • Export Citation
  • 35.

    Kawai A , Arita N , Matsumoto Y , Kawabata M , Chowdhury MS , Saito-Ito A , 2011. Efficacy of chloroquine plus primaquine treatment and pfcrt mutation in uncomplicated falciparum malaria patients in Rangamati, Bangladesh. Parasitol Int 60: 341346.

    • Search Google Scholar
    • Export Citation
  • 36.

    Marma AS , Mita T , Eto H , Tsukahara T , Sarker S , Endo H , 2010. High prevalence of sulfadoxine/pyrimethamine resistance alleles in Plasmodium falciparum parasites from Bangladesh. Parasitol Int 59: 178182.

    • Search Google Scholar
    • Export Citation
  • 37.

    Srimuang K , Miotto O , Lim P , Fairhurst RM , Kwiatkowski DP , Woodrow CJ , Imwong M , 2016. Analysis of anti-malarial resistance markers in pfmdr1 and pfcrt across Southeast Asia in the tracking resistance to artemisinin collaboration. Malar J 15: 541.

    • Search Google Scholar
    • Export Citation
  • 38.

    van den Broek IV , van der Wardt S , Talukder L , Chakma S , Brockman A , Nair S , Anderson TC , 2004. Drug resistance in Plasmodium falciparum from the Chittagong Hill Tracts, Bangladesh. Trop Med Int Health 9: 680687.

    • Search Google Scholar
    • Export Citation
  • 39.

    Akter J , Thriemer K , Khan WA , Sullivan DJ Jr. , Noedl H , Haque R , 2012. Genotyping of Plasmodium falciparum using antigenic polymorphic markers and to study anti-malarial drug resistance markers in malaria endemic areas of Bangladesh. Malar J 11: 386.

    • Search Google Scholar
    • Export Citation
  • 40.

    Snounou G , Singh B , 2002. Nested PCR analysis of Plasmodium parasites. Methods Mol Med 72: 189203.

  • 41.

    Alam MS , Mohon AN , Mustafa S , Khan WA , Islam N , Karim MJ , Khanum H , Sullivan DJ Jr. , Haque R , 2011. Real-time PCR assay and rapid diagnostic tests for the diagnosis of clinically suspected malaria patients in Bangladesh. Malar J 10: 175.

    • Search Google Scholar
    • Export Citation
  • 42.

    Veiga MI , Ferreira PE , Bjorkman A , Gil JP , 2006. Multiplex PCR-RFLP methods for pfcrt, pfmdr1 and pfdhfr mutations in Plasmodium falciparum. Mol Cell Probes 20: 100104.

    • Search Google Scholar
    • Export Citation
  • 43.

    Duraisingh MT , Jones P , Sambou I , von Seidlein L , Pinder M , Warhurst DC , 2000. 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 108: 1323.

    • Search Google Scholar
    • Export Citation
  • 44.

    Ferreira ID , Rosario VE , Cravo PV , 2006. Real-time quantitative PCR with SYBR Green I detection for estimating copy numbers of nine drug resistance candidate genes in Plasmodium falciparum. Malar J 5: 1.

    • Search Google Scholar
    • Export Citation
  • 45.

    WWARN , 2014. Copy Number Estimation of the Plasmodium falciparum Pfmdr1 Gene. Available at: https://www.wwarn.org/tools-resources/procedures. Accessed July 2, 2018.

    • Search Google Scholar
    • Export Citation
  • 46.

    Laufer MK , Thesing PC , Eddington ND , Masonga R , Dzinjalamala FK , Takala SL , Taylor TE , Plowe CV , 2006. Return of chloroquine antimalarial efficacy in Malawi. N Engl J Med 355: 19591966.

    • Search Google Scholar
    • Export Citation
  • 47.

    Mwanza S , Joshi S , Nambozi M , Chileshe J , Malunga P , Kabuya JB , Hachizovu S , Manyando C , Mulenga M , Laufer M , 2016. The return of chloroquine-susceptible Plasmodium falciparum malaria in Zambia. 15: 584.

    • Search Google Scholar
    • Export Citation
  • 48.

    Mohammed A et al. 2013. Trends in chloroquine resistance marker, Pfcrt-K76T mutation ten years after chloroquine withdrawal in Tanzania. Malar J 12: 415.

    • Search Google Scholar
    • Export Citation
  • 49.

    Huang B et al. 2016. Prevalence of crt and mdr-1 mutations in Plasmodium falciparum isolates from Grande Comore island after withdrawal of chloroquine. Malar J 15: 414.

    • Search Google Scholar
    • Export Citation
  • 50.

    Gharbi M et al. 2013. Longitudinal study assessing the return of chloroquine susceptibility of Plasmodium falciparum in isolates from travellers returning from West and Central Africa, 2000-2011. Malar J 12: 35.

    • Search Google Scholar
    • Export Citation
  • 51.

    Awasthi G , Prasad GB , Das A , 2011. Population genetic analyses of Plasmodium falciparum chloroquine receptor transporter gene haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India. Int J Parasitol 41: 705709.

    • Search Google Scholar
    • Export Citation
  • 52.

    Venkatesan M et al. 2014. Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine. Am J Trop Med Hyg 91: 833843.

    • Search Google Scholar
    • Export Citation
  • 53.

    Sisowath C , Strömberg J , Mårtensson A , Msellem M , Obondo C , Björkman A , Gil JP , 2005. In vivo selection of Plasmodium falciparum pfmdr1 86N coding alleles by artemether-lumefantrine (Coartem). J Infect Dis 191: 10141017.

    • Search Google Scholar
    • Export Citation
  • 54.

    Ljolje D et al. 2018. Prevalence of molecular markers of artemisinin and lumefantrine resistance among patients with uncomplicated Plasmodium falciparum malaria in three provinces in Angola, 2015. Malar J 17: 84.

    • Search Google Scholar
    • Export Citation
  • 55.

    Humphreys GS , Merinopoulos I , Ahmed J , Whitty CJ , Mutabingwa TK , Sutherland CJ , Hallett RL , 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: 991997.

    • Search Google Scholar
    • Export Citation
  • 56.

    Baliraine FN , Rosenthal PJ , 2011. Prolonged selection of pfmdr1 polymorphisms after treatment of falciparum malaria with artemether-lumefantrine in Uganda. J Infect Dis 204: 11201124.

    • Search Google Scholar
    • Export Citation
  • 57.

    Happi CT , Gbotosho GO , Folarin OA , Sowunmi A , Hudson T , O'Neil M , Milhous W , Wirth DF , Oduola AM , 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: 888895.

    • Search Google Scholar
    • Export Citation
  • 58.

    Price RN et al. 2004. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet 364: 438447.

  • 59.

    Noedl H , Faiz MA , Yunus EB , Rahman MR , Hossain MA , Samad R , Miller RS , Pang LW , Wongsrichanalai C , 2003. Drug-resistant malaria in Bangladesh: an in vitro assessment. Am J Trop Med Hyg 68: 140142.

    • Search Google Scholar
    • Export Citation
  • 60.

    Ménard D et al. 2016. A worldwide map of Plasmodium falciparum K13-propeller polymorphisms. N Engl J Med 374: 24532464.

Author Notes

Address correspondence to Mohammad Shafiul Alam, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), 68 Shaheed Tajuddin Ahmed Sarani, Dhaka 1212, Bangladesh. E-mail: shafiul@icddrb.org

Financial support: This study was funded by the Swedish International Development Cooperation Agency (SIDA) through the icddr,b core, Swiss Academy of Medical Sciences (SAMS), and Velux Stiftung.

Authors’ addresses: Fatema Tuj Johora, Mohammad Sharif Hossain, Humaira Rashid, Mohammad Golam Kibria, Wasif A. Khan, Rashidul Haque, and Mohammad Shafiul Alam, International Centre for Diarrhoeal Disease Research Bangladesh (icddr,b), Dhaka, Bangladesh, E-mails: fatema.johora@icddrb.org, mshossain@icddrb.org, humaira.rashid@icddrb.org, golam.kibria@icddrb.org, wakhan@icddrb.org, rhaque@icddrb.org, and shafiul@icddrb.org. Rubayet Elahi, Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, E-mail: aelahi3@jhmi.edu. Maisha Khair Nima, Department of Biological Sciences, Boler-Parseghian Center for Rare and Neglected Diseases, University of Notre Dame, Notre Dame, IN, E-mail: mnima@nd.edu. Abu Naser Mohon, Department of Microbiology, Immunology and Infectious Disease, University of Calgary, Alberta, Canada, E-mail: manmohon@ucalgary.ca.

Save