An innovative tool for moving malaria PCR detection of parasite reservoir into the field

Lydie Canier, Nimol Khim, Saorin Kim, Vincent Sluydts, Somony Heng, Dany Dourng, Rotha Eam, Sophy Chy, Chanra Khean, Kaknika Loch, Malen Ken, Hokkean Lim, Sovannaroath Siv, Sochantha Tho, Pascal Masse-Navette, Charlotte Gryseels, Sambunny Uk, Karel Van Roey, Koen Peeters Grietens, Mao Sokny, Boukheng Thavrin, Char Meng Chuor, Vincent Deubel, Lies Durnez, Marc Coosemans, Didier Ménard, Lydie Canier, Nimol Khim, Saorin Kim, Vincent Sluydts, Somony Heng, Dany Dourng, Rotha Eam, Sophy Chy, Chanra Khean, Kaknika Loch, Malen Ken, Hokkean Lim, Sovannaroath Siv, Sochantha Tho, Pascal Masse-Navette, Charlotte Gryseels, Sambunny Uk, Karel Van Roey, Koen Peeters Grietens, Mao Sokny, Boukheng Thavrin, Char Meng Chuor, Vincent Deubel, Lies Durnez, Marc Coosemans, Didier Ménard

Abstract

Background: To achieve the goal of malaria elimination in low transmission areas such as in Cambodia, new, inexpensive, high-throughput diagnostic tools for identifying very low parasite densities in asymptomatic carriers are required. This will enable a switch from passive to active malaria case detection in the field.

Methods: DNA extraction and real-time PCR assays were implemented in an "in-house" designed mobile laboratory allowing implementation of a robust, sensitive and rapid malaria diagnostic strategy in the field. This tool was employed in a survey organized in the context of the MalaResT project (NCT01663831).

Results: The real-time PCR screening and species identification assays were performed in the mobile laboratory between October and November 2012, in Rattanakiri Province, to screen approximately 5,000 individuals in less than four weeks and treat parasite carriers within 24-48 hours after sample collection. An average of 240 clinical samples (and 40 quality control samples) was tested every day, six/seven days per week. Some 97.7% of the results were available <24 hours after the collection. A total of 4.9% were positive for malaria. Plasmodium vivax was present in 61.1% of the positive samples, Plasmodium falciparum in 45.9%, Plasmodium malariae in 7.0% and Plasmodium ovale in 2.0%.

Conclusions: The operational success of this diagnostic set-up proved that molecular testing and subsequent treatment is logistically achievable in field settings. This will allow the detection of clusters of asymptomatic carriers and to provide useful epidemiological information. Fast results will be of great help for staff in the field to track and treat asymptomatic parasitaemic cases. The concept of the mobile laboratory could be extended to other countries for the molecular detection of malaria or other pathogens, or to culture vivax parasites, which does not support long-time delay between sample collection and culture.

Trial registration: ClinicalTrials.gov NCT01555255 NCT01663831.

Figures

Figure 1
Figure 1
Design of the mobile laboratory.
Figure 2
Figure 2
The overview of the process used for high-throughput malaria PCR detection in the field (Panel A), including samples and data management (Panel B).
Figure 3
Figure 3
Linear regression analysis and reproducibility of the real-time PCR screening (Panel A) and melt temperatures (Tm) observed for each Plasmodium species (Panel B).

References

    1. WHO. World Malaria Report 2012. Geneva: World Health Organization; 2012.
    1. Cui L, Yan G, Sattabongkot J, Cao Y, Chen B, Chen X, Fan Q, Fang Q, Jongwutiwes S, Parker D, Sirichaisinthop J, Kyaw MP, Su XZ, Yang H, Yang Z, Wang B, Xu J, Zheng B, Zhong D, Zhou G. Malaria in the Greater Mekong Subregion: heterogeneity and complexity. Acta Trop. 2012;121:227–239. doi: 10.1016/j.actatropica.2011.02.016.
    1. Amaratunga C, Sreng S, Suon S, Phelps ES, Stepniewska K, Lim P, Zhou C, Mao S, Anderson JM, Lindegardh N, Jiang H, Song J, Su XZ, White NJ, Dondorp AM, Anderson TJ, Fay MP, Mu J, Duong S, Fairhurst RM. Artemisinin-resistant Plasmodium falciparum in Pursat province, western Cambodia: a parasite clearance rate study. Lancet Infect Dis. 2012;12:851–858. doi: 10.1016/S1473-3099(12)70181-0.
    1. Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, Lwin KM, Ariey F, Hanpithakpong W, Lee SJ, Ringwald P, Silamut K, Imwong M, Chotivanich K, Lim P, Herdman T, An SS, Yeung S, Singhasivanon P, Day NP, Lindegardh N, Socheat D, White NJ. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2009;361:455–467. doi: 10.1056/NEJMoa0808859.
    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. Phyo AP, Nkhoma S, Stepniewska K, Ashley EA, Nair S, McGready R, Ler Moo C, Al-Saai S, Dondorp AM, Lwin KM, Singhasivanon P, Day NP, White NJ, Anderson TJ, Nosten F. Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. Lancet. 2012;379:1960–1966. doi: 10.1016/S0140-6736(12)60484-X.
    1. Kyaw MP, Nyunt MH, Chit K, Aye MM, Aye KH, Aye MM, Lindegardh N, Tarning J, Imwong M, Jacob CG, Rasmussen C, Perin J, Ringwald P, Nyunt MM. Reduced susceptibility of Plasmodium falciparum to artesunate in southern Myanmar. PLoS One. 2013;8:e57689. doi: 10.1371/journal.pone.0057689.
    1. Tran TH, Nguyen TT, Nguyen HP, Boni MF, Ngo VT, Nguyen TN, Le HT, Cao QT, Pham VT, Phung DT, Le TL, Le TD, Merson L, Dolecek C, Stepniewska K, Ringwald P, White NJ, Farrar J, Wolbers M. In vivo susceptibility of Plasmodium falciparum to artesunate in Binh Phuoc Province Vietnam. Malar J. 2013;11:355.
    1. Gurarie D, Karl S, Zimmerman PA, King CH, St Pierre TG, Davis TM. Mathematical modeling of malaria infection with innate and adaptive immunity in individuals and agent-based communities. PLoS One. 2011;7:e34040.
    1. Incardona S, Vong S, Chiv L, Lim P, Nhem S, Sem R, Khim N, Doung S, Mercereau-Puijalon O, Fandeur T. Large-scale malaria survey in Cambodia: novel insights on species distribution and risk factors. Malar J. 2007;6:37. doi: 10.1186/1475-2875-6-37.
    1. Steenkeste N, Incardona S, Chy S, Duval L, Ekala MT, Lim P, Hewitt S, Sochantha T, Socheat D, Rogier C, Mercereau-Puijalon O, Fandeur T, Ariey F. Towards high- throughput molecular detection of Plasmodium: new approaches and molecular markers. Malar J. 2009;8:86. doi: 10.1186/1475-2875-8-86.
    1. Hoyer S, Nguon S, Kim S, Habib N, Khim N, Sum S, Christophel EM, Bjorge S, Thomson A, Kheng S, Chea N, Yok S, Top S, Ros S, Sophal U, Thompson MM, Mellor S, Ariey F, Witkowski B, Yeang C, Yeung S, Duong S, Newman RD, Menard D. Focused screening and treatment (FSAT): a PCR-based strategy to detect malaria parasite carriers and contain drug resistant P. falciparum, Pailin, Cambodia. PLoS One. 2012;7:e45797. doi: 10.1371/journal.pone.0045797.
    1. Okell LC, Ghani AC, Lyons E, Drakeley CJ. Submicroscopic infection in Plasmodium falciparum-endemic populations: a systematic review and meta-analysis. J Infect Dis. 2009;200:1509–1517. doi: 10.1086/644781.
    1. MalERA Consultative Group on Monitoring Evaluation and Surveillance. A research agenda for malaria eradication: monitoring, evaluation, and surveillance. PLoS Med. 2011;8:e1000400.
    1. Leang R, Barrette A, Bouth DM, Menard D, Abdur R, Duong S, Ringwald P. Efficacy of dihydroartemisinin-piperaquine for treatment of uncomplicated Plasmodium falciparum and Plasmodium vivax in Cambodia, 2008 to 2010. Antimicrob Agents Chemother. 2013;57:818–826. doi: 10.1128/AAC.00686-12.
    1. WHO. Malaria elimination. A field manual for low and moderate endemic countries. Geneva: World Health Organization; 2007.
    1. Rakotonirina H, Barnadas C, Raherijafy R, Andrianantenaina H, Ratsimbasoa A, Randrianasolo L, Jahevitra M, Andriantsoanirina V, Menard D. Accuracy and reliability of malaria diagnostic techniques for guiding febrile outpatient treatment in malaria-endemic countries. Am J Trop Med Hyg. 2008;78:217–221.
    1. Wongsrichanalai C, Barcus MJ, Muth S, Sutamihardja A, Wernsdorfer WH. A review of malaria diagnostic tools: microscopy and rapid diagnostic test (RDT) Am J Trop Med Hyg. 2007;77:119–127.
    1. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, Do Rosario VE, Thaithong S, Brown KN. 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. Singh B, Bobogare A, Cox-Singh J, Snounou G, Abdullah MS, Rahman HA. A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am J Trop Med Hyg. 1999;60:687–692.
    1. Proux S, Suwanarusk R, Barends M, Zwang J, Price RN, Leimanis M, Kiricharoen L, Laochan N, Russell B, Nosten F, Snounou G. Considerations on the use of nucleic acid-based amplification for malaria parasite detection. Malar J. 2011;10:323. doi: 10.1186/1475-2875-10-323.
    1. Maurin M. Real-time PCR as a diagnostic tool for bacterial diseases. Expert Rev Mol Diagn. 2012;12:731–754. doi: 10.1586/erm.12.53.
    1. Waters AP, McCutchan TF. Rapid, sensitive diagnosis of malaria based on ribosomal RNA. Lancet. 1989;1:1343–1346.
    1. Snounou G, Singh B. Nested PCR analysis of Plasmodium parasites. Methods Mol Med. 2002;72:189–203.
    1. Chou M, Kim S, Khim N, Chy S, Sum S, Dourng D, Canier L, Nguon C, Menard D. Performance of “VIKIA Malaria Ag Pf/Pan” (IMACCESS(R)), a new malaria rapid diagnostic test for detection of symptomatic malaria infections. Malar J. 2012;11:295. doi: 10.1186/1475-2875-11-295.
    1. Farrugia C, Cabaret O, Botterel F, Bories C, Foulet F, Costa JM, Bretagne S. Cytochrome b gene quantitative PCR for diagnosing Plasmodium falciparum infection in travelers. J Clin Microbiol. 2011;49:2191–2195. doi: 10.1128/JCM.02156-10.
    1. Haanshuus CG, Mohn SC, Morch K, Langeland N, Blomberg B, Hanevik K. A novel, single-amplification PCR targeting mitochondrial genome highly sensitive and specific in diagnosing malaria among returned travellers in Bergen Norway. Malar J. 2012;12:26.
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    1. FIND. .

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