1921
Volume 97, Issue 2
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645

Abstract

Abstract.

We describe a novel one-step reverse transcriptase real-time PCR (direct RT-PCR) for malaria parasites that amplifies RNA targets directly from blood. We developed the assay to identify gametocyte-specific transcripts in parasites from patient blood samples, as a means of monitoring malaria parasite transmission in field settings. To perform the test, blood is added directly to a master mix in PCR tubes and analyzed by real-time PCR. The limit of detection of the assay on both conventional and portable real-time PCR instruments was 100 parasites/mL for 18S rRNA, and 1,000 parasites/mL for asexual (PFE0065W) and gametocyte (PF14_0367, PFGEXP5) mRNA targets. The usefulness of this assay in field studies was explored in samples from individuals living in a high-transmission region in Cameroon. The sensitivity and specificity of the assay compared with a standard two-step RT-PCR was 100% for 18S rRNA on both conventional and portable instruments. For PF14_0367, the sensitivity and specificity were 85.7% and 70.0%, respectively, on the conventional instrument and 78.6% and 90%, respectively, on the portable instrument. The concordance for assays run on the two instruments was 100% for 18S rRNA, and 79.2% for PF14_0367, with most discrepancies resulting from samples with low transcript levels. The results show asexual and sexual stage RNA targets can be detected directly from blood samples in a simple one-step test on a field-friendly instrument. This assay may be useful for monitoring malaria parasite transmission potential in elimination settings, where sensitive diagnostics are needed to evaluate the progress of malaria eradication initiatives.

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2017-08-02
2018-09-24
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References

  1. Mendis K, , Rietveld A, Warsame M, Bosman A, Greenwood B, Wernsdorfer WH, 2009. From malaria control to eradication: the WHO perspective. Trop Med Int Health 14: 802809.[Crossref]
  2. World Health Organization, 2016. World Health Organization: World Malaria Report. Geneva, Switzerland: World Health Organization.
  3. Alonso PL, , 2011. A research agenda to underpin malaria eradication. PLoS Med 8: e1000406.[Crossref]
  4. Churcher TS, Trape JF, Cohuet A, , 2015. Human-to-mosquito transmission efficiency increases as malaria is controlled. Nat Commun 6: 6054.[Crossref]
  5. Okell LC, , Bousema T, Griffin JT, Ouédraogo AL, Ghani AC, Drakeley CJ, 2012. Factors determining the occurrence of submicroscopic malaria infections and their relevance for control. Nat Commun 3: 1237.[Crossref]
  6. Bousema T, Okell LC, , Felger I, Drakeley C, 2014. Asymptomatic malaria infections: detectability, transmissibility and public health relevance. Nat Rev Microbiol 12: 833–40.
  7. Mwingira F, Genton B, Kabanywanyi AN, Felger I, , 2014. Comparison of detection methods to estimate asexual Plasmodium falciparum parasite prevalence and gametocyte carriage in a community survey in Tanzania. Malar J 13: 433.[Crossref]
  8. Britton S, Cheng Q, McCarthy JS, , 2016. Novel molecular diagnostic tools for malaria elimination: a review of options from the point of view of high-throughput and applicability in resource limited settings. Malar J 15: 88.[Crossref]
  9. Slater HC, , 2015. Assessing the impact of next-generation rapid diagnostic tests on Plasmodium falciparum malaria elimination strategies. Nature 528: S94S101.[Crossref]
  10. Goncalves BP, Drakeley C, Bousema T, , 2016. Infectivity of microscopic and submicroscopic malaria parasite infections in areas of low malaria endemicity. J Infect Dis 213: 15161517.[Crossref]
  11. Lin JT, , 2015. Microscopic Plasmodium falciparum gametocytemia and infectivity to mosquitoes in Cambodia. J Infect Dis 213: 14911494.[Crossref]
  12. Stone W, Goncalves BP, Bousema T, Drakeley C, , 2015. Assessing the infectious reservoir of falciparum malaria: past and future. Trends Parasitol 31: 287296.[Crossref]
  13. Bousema T, Drakeley C, , 2011. Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination. Clin Microbiol Rev 24: 377410.[Crossref]
  14. Babiker HA, Schneider P, Reece SE, , 2008. Gametocytes: insights gained during a decade of molecular monitoring. Trends Parasitol 24: 525530.[Crossref]
  15. Schneider P, , Schoone G, Schallig H, Verhage D, Telgt D, Eling W, Sauerwein R, 2004. Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification. Mol Biochem Parasitol 137: 3541.[Crossref]
  16. Buates S, , Bantuchai S, Sattabongkot J, Han ET, Tsuboi T, Udomsangpetch R, Sirichaisinthop J, Tan-ariya P, 2010. Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for clinical detection of Plasmodium falciparum gametocytes. Parasitol Int 59: 414420.[Crossref]
  17. Babiker HA, , Abdel-Wahab A, Ahmed S, Suleiman S, Ranford-Cartwright L, Carter R, Walliker D, 1999. Detection of low level Plasmodium falciparum gametocytes using reverse transcriptase polymerase chain reaction. Mol Biochem Parasitol 99: 143148.[Crossref]
  18. Koepfli C, , 2015. Blood-stage parasitaemia and age determine Plasmodium falciparum and P. vivax gametocytaemia in Papua New Guinea. PLoS One 10: e0126747.[Crossref]
  19. Wampfler R, , Timinao L, Beck HP, Soulama I, Tiono AB, Siba P, Mueller I, Felger I, 2014. Novel genotyping tools for investigating transmission dynamics of Plasmodium falciparum . J Infect Dis 210: 11881197.[Crossref]
  20. Alano P, , 1995. COS cell expression cloning of Pfg377, a Plasmodium falciparum gametocyte antigen associated with osmiophilic bodies. Mol Biochem Parasitol 74: 143156.[Crossref]
  21. Schneider P, , Wolters L, Schoone G, Schallig H, Sillekens P, Hermsen R, Sauerwein R, 2005. Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum . J Clin Microbiol 43: 402405.[Crossref]
  22. Wampfler R, , Mwingira F, Javati S, Robinson L, Betuela I, Siba P, Beck HP, Mueller I, Felger I, 2013. Strategies for detection of Plasmodium species gametocytes. PLoS One 8: e76316.[Crossref]
  23. Taylor BJ, , Martin KA, Arango E, Agudelo OM, Maestre A, Yanow SK, 2011. Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples. Malar J 10: 244.[Crossref]
  24. Taylor BJ, , 2014. A lab-on-chip for malaria diagnosis and surveillance. Malar J 13: 179.[Crossref]
  25. Joice R, , 2013. Inferring developmental stage composition from gene expression in human malaria. PLOS Comput Biol 9: e1003392.[Crossref]
  26. Trager W, Jensen JB, , 1976. Human malaria parasites in continuous culture. Science 193: 673675.[Crossref]
  27. Ifediba T, Vanderberg JP, , 1981. Complete in vitro maturation of Plasmodium falciparum gametocytes. Nature 294: 364366.[Crossref]
  28. Ponnudurai T, Lensen AH, Leeuwenberg AD, Meuwissen JH, , 1982. Cultivation of fertile Plasmodium falciparum gametocytes in semi-automated systems. 1. Static cultures. Trans R Soc Trop Med Hyg 76: 812818.[Crossref]
  29. Ponnudurai T, , Lensen AH, Van Gemert GJ, Bensink MP, Bolmer M, Meuwissen JH, 1989. Infectivity of cultured Plasmodium falciparum gametocytes to mosquitoes. Parasitology 98: 165173.[Crossref]
  30. Ponnudurai T, Lensen AH, Meis JF, Meuwissen JH, , 1986. Synchronization of Plasmodium falciparum gametocytes using an automated suspension culture system. Parasitology 93: 263274.[Crossref]
  31. van Schaijk BC, , 2008. Gene disruption of Plasmodium falciparum p52 results in attenuation of malaria liver stage development in cultured primary human hepatocytes. PLoS One 3: e3549.[Crossref]
  32. Sandeu MM, , Abate L, Tchioffo MT, Bayibéki AN, Awono-Ambéné PH, Nsango SE, Chesnais CB, Dinglasan RR, de Meeûs T, Morlais I, 2016. Impact of exposure to mosquito transmission-blocking antibodies on Plasmodium falciparum population genetic structure. Infect Genet Evol 45: 138144.[Crossref]
  33. Kamau E, , Tolbert LS, Kortepeter L, Pratt M, Nyakoe N, Muringo L, Ogutu B, Waitumbi JN, Ockenhouse CF, 2011. Development of a highly sensitive genus-specific quantitative reverse transcriptase real-time PCR assay for detection and quantitation of Plasmodium by amplifying RNA and DNA of the 18S rRNA genes. J Clin Microbiol 49: 29462953.[Crossref]
  34. Mercereau-Puijalon O, Barale JC, Bischoff E, , 2002. Three multigene families in Plasmodium parasites: facts and questions. Int J Parasitol 32: 13231344.[Crossref]
  35. Murphy SC, , 2012. Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials. Am J Trop Med Hyg 86: 383394.[Crossref]
  36. Wongsrichanalai C, Barcus MJ, Muth S, Sutamihardja A, Wernsdorfer WH, , 2007. A review of malaria diagnostic tools: microscopy and rapid diagnostic test (RDT). Am J Trop Med Hyg 77: 119127.
  37. Blisnick T, , Morales Betoulle ME, Barale JC, Uzureau P, Berry L, Desroses S, Fujioka H, Mattei D, Braun Breton C, 2000. Pfsbp1, a Maurer’s cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton. Mol Biochem Parasitol 111: 107121.[Crossref]
  38. Tiburcio M, , Dixon MW, Looker O, Younis SY, Tilley L, Alano P, 2015. Specific expression and export of the Plasmodium falciparum Gametocyte EXported Protein-5 marks the gametocyte ring stage. Malar J 14: 334.[Crossref]
  39. World Health Organization, 2015. Global Technical Strategy for Malaria 2016–2030. Geneva, Switzerland: World Health Organization.
  40. Malaria Policy Advisory Committee, 2014. WHO Evidence Review Group on Malaria Diagnosis in Low Transmission Settings. Geneva, Switzerland: World Health Organization.
  41. Hopkins H, , 2013. Highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop-mediated isothermal amplification kit in a remote clinic in Uganda. J Infect Dis 208: 645652.[Crossref]
  42. Tsui NB, Ng EK, Lo YM, , 2002. Stability of endogenous and added RNA in blood specimens, serum, and plasma. Clin Chem 48: 16471653.
  43. Al-Soud WA, Radstrom P, , 2001. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 39: 485493.[Crossref]
  44. Nikolaeva D, Draper SJ, Biswas S, , 2015. Toward the development of effective transmission-blocking vaccines for malaria. Expert Rev Vaccines 14: 653680.[Crossref]
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Supplementary Data

Supplemental Figure and Table

  • Received : 16 Jan 2017
  • Accepted : 06 Apr 2017

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