• 1

    Yeung S, Pongtavornpinyo W, Hastings IM, Mills AJ, White NJ, 2004. Antimalarial drug resistance, artemisinin-based combination therapy, and the contribution of modeling to elucidating policy choices. Am J Trop Med Hyg 71 :179–186.

    • Search Google Scholar
    • Export Citation
  • 2

    Rieckmann KH, Campbell GH, Sax LJ, Mrema JE, 1978. Drug sensitivity of Plasmodium falciparum. An in-vitro microtechnique. Lancet 1 :22–23.

    • Search Google Scholar
    • Export Citation
  • 3

    Desjardins RE, Canfield CJ, Haynes JD, Chulay JD, 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16 :710–718.

    • Search Google Scholar
    • Export Citation
  • 4

    Makler MT, Ries JM, Williams JA, Bancroft JE, Piper RC, Gibbins BL, Hinrichs DJ, 1993. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. Am J Trop Med Hyg 48 :739–741.

    • Search Google Scholar
    • Export Citation
  • 5

    Noedl H, Wernsdorfer WH, Miller RS, Wongsrichanalai C, 2002. Histidine-rich protein II: a novel approach to malaria drug sensitivity testing. Antimicrob Agents Chemother 46 :1658–1664.

    • Search Google Scholar
    • Export Citation
  • 6

    Noedl H, Bronnert J, Yingyuen K, Attlmayr B, Kollaritsch H, Fukuda M, 2005. Simple histidine-rich protein 2 double-site sandwich enzyme-linked immunosorbent assay for use in malaria drug sensitivity testing. Antimicrob Agents Chemother 49 :3575–3577.

    • Search Google Scholar
    • Export Citation
  • 7

    Druilhe P, Moreno A, Blanc C, Brasseur PH, Jacquier P, 2001. A colorimetric in vitro drug sensitivity assay for Plasmodium falciparum based on a highly sensitive double-site lactate dehydrogenase antigen-capture enzyme-linked immunosorbent assay. Am J Trop Med Hyg 64 :233–241.

    • Search Google Scholar
    • Export Citation
  • 8

    Bennett TN, Paguio M, Gligorijevic B, Seudieu C, Kosar AD, Davidson E, Roepe PD, 2004. Novel, rapid, and inexpensive cell-based quantification of antimalarial drug efficacy. Antimicrob Agents Chemother 48 :1807–1810.

    • Search Google Scholar
    • Export Citation
  • 9

    Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M, 2004. Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrob Agents Chemother 48 :1803–1806.

    • Search Google Scholar
    • Export Citation
  • 10

    Bacon DJ, Latour C, Lucas C, Colina O, Ringwald P, Picot S, 2007. Comparison of a SYBR Green I based assay with an HRPII ELISA method for in vitro antimalarial drug efficacy testing and application to clinical isolates. Antimicrob Agents Chemother 51 :1172–1178.

    • Search Google Scholar
    • Export Citation
  • 11

    Baniecki ML, Wirth DF, Clardy J, 2007. High-throughput Plasmodium falciparum growth assay for malaria drug discovery. Antimicrob Agents Chemother 51 :716–723.

    • Search Google Scholar
    • Export Citation
  • 12

    Johnson JD, Dennull RA, Gerena L, Lopez-Sanchez M, Roncal NE, Waters NC, 2007. Assessment and continued validation of the malaria SYBR green I-based fluorescence assay for use in malaria drug screening. Antimicrob Agents Chemother 51 :1926–1933.

    • Search Google Scholar
    • Export Citation
  • 13

    Wellems TE, Panton LJ, Gluzman IY, do Rosario VE, Gwadz RW, Walker-Jonah A, Krogstad DJ, 1990. Chloroquine resistance not linked to mdr-like genes in a Plasmodium falciparum cross. Nature 345 :253–255.

    • Search Google Scholar
    • Export Citation
  • 14

    Wongsrichanalai C, Lin K, Pang LW, Faiz MA, Noedl H, Wimonwattrawatee T, Laoboonchai A, Kawamoto F, 2001. In vitro susceptibility of Plasmodium falciparum isolates from Myanmar to antimalarial drugs. Am J Trop Med Hyg 65 :450–455.

    • Search Google Scholar
    • Export Citation
  • 15

    Noedl H, Wongsrichanalai C, Wernsdorfer WH, 2003. Malaria drug-sensitivity testing: new assays, new perspectives. Trends Parasitol 19 :175–181.

    • Search Google Scholar
    • Export Citation
  • 16

    Ferdig MT, Cooper RA, Mu J, Deng B, Joy DA, Su X-z, Wellems TE, 2004. Dissecting the loci of low-level quinine resistance in malaria parasites. Mol Microbiol 52 :985–997.

    • Search Google Scholar
    • Export Citation
  • 17

    Cooper RA, Lane KD, Deng B, Mu J, Patel JJ, Wellems TE, Su X-z, Ferdig MT, 2007. Mutations in transmembrane domains 1, 4 and 9 of the Plasmodium falciparum chloroquine resistance transporter alter susceptibility to chloroquine, quinine and quinidine. Mol Microbiol 63 :270–282.

    • Search Google Scholar
    • Export Citation
  • 18

    Anderson TJ, Haubold B, Williams JT, Estrada-Franco Section Sign JG, Richardson L, Mollinedo R, Bockarie M, Mokili J, Mharakurwa S, French N, Whitworth J, Velez ID, Brockman AH, Nosten F, Ferreira MU, Day KP, 2000. Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum. Mol Biol Evol 17 :1467–1482.

    • Search Google Scholar
    • Export Citation
  • 19

    Bendixen M, Msangeni HA, Pedersen BV, Shayo D, Bodker R, 2001. Diversity of Plasmodium falciparum populations and complexity of infections in relation to transmission intensity and host age: a study from the Usambara Mountains, Tanzania. Trans R Soc Trop Med Hyg 95 :143–148.

    • Search Google Scholar
    • Export Citation
  • 20

    Kobbe R, Neuhoff R, Marks F, Adjei S, Langefeld I, von Reden C, Adjei O, Meyer CG, May J, 2006. Seasonal variation and high multiplicity of first Plasmodium falciparum infections in children from a holoendemic area in Ghana, West Africa. Trop Med Int Health 11 :613–619.

    • Search Google Scholar
    • Export Citation
  • 21

    Lee SA, Yeka A, Nsobya SL, Dokomajilar C, Rosenthal PJ, Talisuna A, Dorsey G, 2006. Complexity of Plasmodium falciparum infections and antimalarial drug efficacy at 7 sites in Uganda. J Infect Dis 193 :1160–1163.

    • Search Google Scholar
    • Export Citation
  • 22

    Viriyakosol S, Siripoon N, Petcharapirat C, Petcharapirat P, Jarra W, Thaithong S, Brown KN, Snounou G, 1995. Genotyping of Plasmodium falciparum isolates by the polymerase chain reaction and potential uses in epidemiological studies. Bull World Health Organ 73 :85–95.

    • Search Google Scholar
    • Export Citation
  • 23

    Babiker H, Ranford-Cartwright L, Sultan A, Satti G, Walliker D, 1994. Genetic evidence that RI chloroquine resistance of Plasmodium falciparum is caused by recrudescence of resistant parasites. Trans R Soc Trop Med Hyg 88 :328–331.

    • Search Google Scholar
    • Export Citation
  • 24

    Ferdig MT, Su X-z, 2000. Microsatellite markers and genetic mapping in Plasmodium falciparum. Parasitol Today 16 :307–312.

  • 25

    Anderson TJ, Nair S, Qin H, Singlam S, Brockman A, Paiphun L, Nosten F, 2005. Are transporter genes other than the chloroquine resistance locus (pfcrt) and multidrug resistance gene (pfmdr) associated with antimalarial drug resistance? Antimicrob Agents Chemother 49 :2180–2188.

    • Search Google Scholar
    • Export Citation
  • 26

    Nair S, Nash D, Sudimack D, Jaidee A, Barends M, Uhlemann AC, Krishna S, Nosten F, Anderson TJ, 2007. Recurrent gene amplification and soft selective sweeps during evolution of multidrug resistance in malaria parasites. Mol Biol Evol 24 :562–573.

    • Search Google Scholar
    • Export Citation
  • 27

    Dolan SA, Herrfeldt JA, Wellems TE, 1993. Restriction polymorphisms and fingerprint patterns from an interspersed repetitive element of Plasmodium falciparum DNA. Mol Biochem Parasitol 61 :137–142.

    • Search Google Scholar
    • Export Citation
  • 28

    Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193 :673–675.

  • 29

    Su X-z, Ferdig MT, Huang Y, Huynh CQ, Liu A, You J, Wootton JC, Wellems TE, 1999. A genetic map and recombination parameters of the human malaria parasite Plasmodium falciparum. Science 286 :1351–1353.

    • Search Google Scholar
    • Export Citation
  • 30

    Mu J, Ferdig MT, Feng X, Joy DA, Duan J, Furuya T, Subramanian G, Aravind L, Cooper RA, Wootton JC, Xiong M, Su X-z, 2003. Multiple transporters associated with malaria parasite responses to chloroquine and quinine. Mol Microbiol 49 :977–989.

    • Search Google Scholar
    • Export Citation
  • 31

    Cheesman SJ, de Roode JC, Read AF, Carter R, 2003. Real-time quantitative PCR for analysis of genetically mixed infections of malaria parasites: technique validation and applications. Mol Biochem Parasitol 131 :83–91.

    • Search Google Scholar
    • Export Citation
  • 32

    McNamara DT, Thomson JM, Kasehagen LJ, Zimmerman PA, 2004. Development of a multiplex PCR-ligase detection reaction assay for diagnosis of infection by the four parasite species causing malaria in humans. J Clin Microbiol 42 :2403–2410.

    • Search Google Scholar
    • Export Citation
  • 33

    Brockman A, Paul RE, Anderson TJ, Hackford I, Phaiphun L, Looareesuwan S, Nosten F, Day KP, 1999. Application of genetic markers to the identification of recrudescent Plasmodium falciparum infections on the northwestern border of Thailand. Am J Trop Med Hyg 60 :14–21.

    • Search Google Scholar
    • Export Citation
  • 34

    Nyachieo A, VAN Omervier C, Laurent T, Dujardin JC, D’Alessandro U, 2005. Plasmodium falciparum genotyping by microsatellites as a method to distinguish between recrudescent and new infections. Am J Trop Med Hyg 73 :210–213.

    • Search Google Scholar
    • Export Citation
  • 35

    Colborn JM, Koita OA, Cisse O, Bagayoko MW, Guthrie EJ, Krogstad DJ, 2006. Identifying and quantifying genotypes in polyclonal infections due to single species. Emerg Infect Dis 12 :475–482.

    • Search Google Scholar
    • Export Citation
  • 36

    Greenhouse B, Myrick A, Dokomajilar C, Woo JM, Carlson EJ, Rosenthal PJ, Dorsey G, 2006. Validation of microsatellite markers for use in genotyping polyclonal Plasmodium falciparum infections. Am J Trop Med Hyg 75 :836–842.

    • Search Google Scholar
    • Export Citation
  • 37

    Certain LK, Sibley CH, 2007. Plasmodium falciparum: a novel method for analyzing haplotypes in mixed infections. Exp Parasitol 115 :233–241.

    • Search Google Scholar
    • Export Citation
  • 38

    Takala SL, Smith DL, Stine OC, Coulibaly D, Thera MA, Doumbo OK, Plowe CV, 2006. A high-throughput method for quantifying alleles and haplotypes of the malaria vaccine candidate Plasmodium falciparum merozoite surface protein-1 19 kDa. Malar J 5 :31.

    • Search Google Scholar
    • Export Citation
  • 39

    Cojean S, Noel A, Garnier D, Hubert V, Le Bras J, Durand R, 2006. Lack of association between putative transporter gene polymorphisms in Plasmodium falciparum and chloroquine resistance in imported malaria isolates from Africa. Malar J 5 :24.

    • Search Google Scholar
    • Export Citation
  • 40

    Martinelli A, Hunt P, Cheesman SJ, Carter R, 2004. Amplified fragment length polymorphism measures proportions of malaria parasites carrying specific alleles in complex genetic mixtures. Mol Biochem Parasitol 136 :117–122.

    • Search Google Scholar
    • Export Citation
  • 41

    Hunt P, Fawcett R, Carter R, Walliker D, 2005. Estimating SNP proportions in populations of malaria parasites by sequencing: validation and applications. Mol Biochem Parasitol 143 :173–182.

    • Search Google Scholar
    • Export Citation
  • 42

    Cheesman S, Creasey A, Degnan K, Kooij T, Afonso A, Cravo P, Carter R, Hunt P, 2007. Validation of pyrosequencing for accurate and high throughput estimation of allele frequencies in malaria parasites. Mol Biochem Parasitol 152 :213–219.

    • Search Google Scholar
    • Export Citation
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

 

 

Effects of Plasmodium falciparum Mixed Infections on In Vitro Antimalarial Drug Tests and Genotyping

View More View Less
  • 1 Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland; Key Laboratory of the Ministry of Education for Cell Biology and Tumour Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, Fujian, The People’s Republic of China
Restricted access

Studying drug resistance in Plasmodium falciparum requires accurate measurement of parasite response to a drug. Factors such as mixed infection of drug-resistant and -sensitive parasites can influence drug test outcome. Polymorphic DNA sequences are frequently used to detect mixed infections; infections with a single genotype or having a minor allele smaller than a subjectively selected cut-off value are often considered single infection. We studied the effects of mixed parasite populations containing various ratios of parasites resistant and sensitive to chloroquine on outcomes of drug tests and how ratios of parasite mixtures correlated with genotypes using polymerase chain reaction–based methods. Our results show that a mixture with a resistant population as low as 10% could greatly impact a drug test outcome. None of the genotyping methods could reliably detect minor DNA alleles at ≤ 10%. Mixed infection presents a serious problem for drug tests, and genotyping using microsatellite or other methods may not reliably reflect true ratios of alleles.

Author Notes

Reprint requests: Xin-zhuan Su, Laboratory of Malaria and Vector Research, National Institutes of Health, 12735 Twinbrook Parkway, Room 3E24B, Rockville, MD 20852, E-mail: xsu@niaid.nih.gov.
Save