Critical Evaluation of Molecular Monitoring in Malaria Drug Efficacy Trials and Pitfalls of Length-Polymorphic Markers

Overview

Journal Antimicrob Agents Chemother

Publisher American Society for Microbiology

Specialty Pharmacology

Date 2016 Nov 9

PMID 27821442

Citations 37

Authors

Camilla Messerli

Natalie E Hofmann

Hans-Peter Beck

Ingrid Felger

Affiliations

Soon will be listed here.

Abstract

Estimation of drug efficacy in antimalarial drug trials requires parasite genotyping to distinguish new infections from treatment failures. When using length-polymorphic molecular markers, preferential amplification of short fragments can compromise detection of coinfections, potentially leading to misclassification of treatment outcome. We quantified minority clone detectability and competition among msp1, msp2, and glurp amplicons using mixtures of Plasmodium falciparum strains and investigated the impact of template competition on genotyping outcomes in 44 paired field samples. Substantial amplification bias was detected for all three markers, with shorter fragments outperforming larger fragments. The strongest template competition was observed for the marker glurp Detection of glurp fragments in multiclonal infections was severely compromised. Eight of 44 sample pairs were identified as new infections by all three markers. Ten pairs were defined as new infections based on one marker alone, seven of which were defined by the questionable marker glurp The impact of size-dependent template competition on genotyping outcomes therefore calls for necessary amendments to the current WHO recommendations for PCR correction of malaria drug trial endpoints. Accuracy of genotyping outcomes could be improved by separate amplification reactions per allelic family and basing results on markers msp1 and msp2 first, with glurp only used to resolve discordant results.

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References

Hofmann N, Mwingira F, Shekalaghe S, Robinson L, Mueller I, Felger I . Ultra-sensitive detection of Plasmodium falciparum by amplification of multi-copy subtelomeric targets. PLoS Med. 2015; 12(3):e1001788. PMC: 4348198. DOI: 10.1371/journal.pmed.1001788. View

Neafsey D, Juraska M, Bedford T, Benkeser D, Valim C, Griggs A . Genetic Diversity and Protective Efficacy of the RTS,S/AS01 Malaria Vaccine. N Engl J Med. 2015; 373(21):2025-2037. PMC: 4762279. DOI: 10.1056/NEJMoa1505819. View

Duru K, Thomas B . Genetic Diversity and Allelic Frequency of Glutamate-Rich Protein (GLURP) in Plasmodium falciparum Isolates from Sub-Saharan Africa. Microbiol Insights. 2014; 7:35-9. PMC: 4240185. DOI: 10.4137/MBI.S20618. View

Liljander A, Wiklund L, Falk N, Kweku M, Martensson A, Felger I . Optimization and validation of multi-coloured capillary electrophoresis for genotyping of Plasmodium falciparum merozoite surface proteins (msp1 and 2). Malar J. 2009; 8:78. PMC: 2680902. DOI: 10.1186/1475-2875-8-78. View

Gupta V, Dorsey G, Hubbard A, Rosenthal P, Greenhouse B . Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda. Malar J. 2010; 9:19. PMC: 2817701. DOI: 10.1186/1475-2875-9-19. View

Koepfli C, Schoepflin S, Bretscher M, Lin E, Kiniboro B, Zimmerman P . How much remains undetected? Probability of molecular detection of human Plasmodia in the field. PLoS One. 2011; 6(4):e19010. PMC: 3084249. DOI: 10.1371/journal.pone.0019010. View

Felger I, Beck H . Genotyping of Plasmodium falciparum. PCR-RFLP analysis. Methods Mol Med. 2002; 72:117-29. DOI: 10.1385/1-59259-271-6:117. View

Snounou G . Genotyping of Plasmodium spp. Nested PCR. Methods Mol Med. 2002; 72:103-16. DOI: 10.1385/1-59259-271-6:103. View

Snounou G, Beck H . The use of PCR genotyping in the assessment of recrudescence or reinfection after antimalarial drug treatment. Parasitol Today. 2006; 14(11):462-7. DOI: 10.1016/s0169-4758(98)01340-4. View

10.

Felger I, Maire M, Bretscher M, Falk N, Tiaden A, Sama W . The dynamics of natural Plasmodium falciparum infections. PLoS One. 2012; 7(9):e45542. PMC: 3445515. DOI: 10.1371/journal.pone.0045542. View

11.

Beck H, Felger I, Huber W, Steiger S, Smith T, Weiss N . Analysis of multiple Plasmodium falciparum infections in Tanzanian children during the phase III trial of the malaria vaccine SPf66. J Infect Dis. 1997; 175(4):921-6. DOI: 10.1086/513991. View

12.

Mwingira F, Nkwengulila G, Schoepflin S, Sumari D, Beck H, Snounou G . Plasmodium falciparum msp1, msp2 and glurp allele frequency and diversity in sub-Saharan Africa. Malar J. 2011; 10:79. PMC: 3090371. DOI: 10.1186/1475-2875-10-79. View

13.

Snounou G, Zhu X, Siripoon N, Jarra W, Thaithong S, BROWN K . Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand. Trans R Soc Trop Med Hyg. 2000; 93(4):369-74. DOI: 10.1016/s0035-9203(99)90120-7. View

14.

Smith T, Felger I, Fraser-Hurt N, Beck H . Effect of insecticide-treated bed nets on the dynamics of multiple Plasmodium falciparum infections. Trans R Soc Trop Med Hyg. 1999; 93 Suppl 1:53-7. DOI: 10.1016/s0035-9203(99)90328-0. View

15.

Mueller I, Schoepflin S, Smith T, Benton K, Bretscher M, Lin E . Force of infection is key to understanding the epidemiology of Plasmodium falciparum malaria in Papua New Guinean children. Proc Natl Acad Sci U S A. 2012; 109(25):10030-5. PMC: 3382533. DOI: 10.1073/pnas.1200841109. View

16.

Rosanas-Urgell A, Mueller D, Betuela I, Barnadas C, Iga J, Zimmerman P . Comparison of diagnostic methods for the detection and quantification of the four sympatric Plasmodium species in field samples from Papua New Guinea. Malar J. 2010; 9:361. PMC: 3016373. DOI: 10.1186/1475-2875-9-361. View

17.

de Souza A, de Araujo F, Fontes C, Carvalho L, de Brito C, de Sousa T . Multiple-clone infections of Plasmodium vivax: definition of a panel of markers for molecular epidemiology. Malar J. 2015; 14:330. PMC: 4548710. DOI: 10.1186/s12936-015-0846-5. View

18.

Falk N, Maire N, Sama W, Owusu-Agyei S, Smith T, Beck H . Comparison of PCR-RFLP and Genescan-based genotyping for analyzing infection dynamics of Plasmodium falciparum. Am J Trop Med Hyg. 2006; 74(6):944-50. View

19.

Daniels R, Volkman S, Milner D, Mahesh N, Neafsey D, Park D . A general SNP-based molecular barcode for Plasmodium falciparum identification and tracking. Malar J. 2008; 7:223. PMC: 2584654. DOI: 10.1186/1475-2875-7-223. View

20.

Bretscher M, Valsangiacomo F, Owusu-Agyei S, Penny M, Felger I, Smith T . Detectability of Plasmodium falciparum clones. Malar J. 2010; 9:234. PMC: 2936403. DOI: 10.1186/1475-2875-9-234. View