Loop mediated isothermal amplification (LAMP) accurately detects malaria DNA from filter paper blood samples of low density parasitaemias

Berit Aydin-Schmidt, Weiping Xu, Iveth J González, Spencer D Polley, David Bell, Delér Shakely, Mwinyi I Msellem, Anders Björkman, Andreas Mårtensson, Berit Aydin-Schmidt, Weiping Xu, Iveth J González, Spencer D Polley, David Bell, Delér Shakely, Mwinyi I Msellem, Anders Björkman, Andreas Mårtensson

Abstract

Background: Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots.

Methods and findings: Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6-782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94-99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0-4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1-98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2-99.9) and 76.9% (95%CI 46.2-95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1-100) in both study groups.

Conclusion: Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low-density malaria infections in pre-elimination settings.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Flow chart of study. Reference…
Figure 1. Flow chart of study. Reference standard
= Cytochrome B nested PCR. Gold standard = Cytochrome B real-time PCR corrected nested PCR.

References

    1. Moonen B, Cohen JM, Snow RW, Slutsker L, Drakeley C, et al. (2010) Operational strategies to achieve and maintain malaria elimination. Lancet 376: 1592–1603.
    1. A research agenda for malaria eradication: Diagnoses and diagnostics. PLoS medicine 8: e1000396.
    1. Manjurano A, Okell L, Lukindo T, Reyburn H, Olomi R, et al. (2011) Association of sub-microscopic malaria parasite carriage with transmission intensity in north-eastern Tanzania. Malaria journal 10: 370.
    1. Okell LC, Bousema T, Griffin JT, Ouedraogo AL, Ghani AC, et al. (2012) Factors determining the occurrence of submicroscopic malaria infections and their relevance for control. Nature communications 3: 1237.
    1. Hopkins H, Gonzalez IJ, Polley SD, Angutoko P, Ategeka J, et al. (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. The Journal of infectious diseases
    1. Mills CD, Burgess DC, Taylor HJ, Kain KC (1999) Evaluation of a rapid and inexpensive dipstick assay for the diagnosis of Plasmodium falciparum malaria. Bulletin of the World Health Organization 77: 553–559.
    1. McMorrow ML, Aidoo M, Kachur SP (2011) Malaria rapid diagnostic tests in elimination settings–can they find the last parasite? Clinical microbiology and infection: the official publication of the European Society of Clinical Microbiology and Infectious Diseases 17: 1624–1631.
    1. Hanscheid T, Grobusch MP (2002) How useful is PCR in the diagnosis of malaria? Trends in parasitology 18: 395–398.
    1. Njiru ZK (2012) Loop-mediated isothermal amplification technology: towards point of care diagnostics. PLoS neglected tropical diseases 6: e1572.
    1. Han ET, Watanabe R, Sattabongkot J, Khuntirat B, Sirichaisinthop J, et al. (2007) Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis. Journal of clinical microbiology 45: 2521–2528.
    1. Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, et al. (2006) Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clinical chemistry 52: 303–306.
    1. Polley SD, Mori Y, Watson J, Perkins MD, Gonzalez IJ, et al. (2010) Mitochondrial DNA targets increase sensitivity of malaria detection using loop-mediated isothermal amplification. Journal of clinical microbiology 48: 2866–2871.
    1. Bossuyt PM, Reitsma JB, Bruns DE, Gatsonis CA, Glasziou PP, et al. (2003) Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD initiative. Clinical chemistry and laboratory medicine: CCLM/FESCC 41: 68–73.
    1. Shakely D, Elfving K, Aydin-Schmidt B, Msellem MI, Morris U, et al. (2013) The usefulness of rapid diagnostic tests in the new context of low malaria transmission in Zanzibar. PloS one 8: e72912.
    1. Shokoples SE, Ndao M, Kowalewska-Grochowska K, Yanow SK (2009) Multiplexed real-time PCR assay for discrimination of Plasmodium species with improved sensitivity for mixed infections. Journal of clinical microbiology 47: 975–980.
    1. Steenkeste N, Incardona S, Chy S, Duval L, Ekala MT, et al. (2009) Towards high-throughput molecular detection of Plasmodium: new approaches and molecular markers. Malaria journal 8: 86.
    1. Dahlstrom S, Veiga MI, Ferreira P, Martensson A, Kaneko A, et al. (2008) Diversity of the sarco/endoplasmic reticulum Ca(2+)-ATPase orthologue of Plasmodium falciparum (PfATP6). Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 8: 340–345.
    1. Wooden J, Kyes S, Sibley CH (1993) PCR and strain identification in Plasmodium falciparum. Parasitology today 9: 303–305.
    1. Hsiang MS, Lin M, Dokomajilar C, Kemere J, Pilcher CD, et al. (2010) PCR-based pooling of dried blood spots for detection of malaria parasites: optimization and application to a cohort of Ugandan children. Journal of clinical microbiology 48: 3539–3543.
    1. Kamau E, Alemayehu S, Feghali KC, Saunders D, Ockenhouse CF (2013) Multiplex qPCR for detection and absolute quantification of malaria. PloS one 8: e71539.
    1. Foundation for innovative new diagnostics FIND (2012) Manual of Standard Operating Procedures for malaria LAMP. Geneva, Tokyo, London.
    1. Polley SD, Gonzalez IJ, Mohamed D, Daly R, Bowers K, et al. (2013) Clinical Evaluation of a Loop-Mediated Amplification Kit for Diagnosis of Imported Malaria. The Journal of infectious diseases
    1. Helsinki WMADo (2008; ICH-GCP, 1996) Ethical Principles for Medical Research Involving Human Subjects.
    1. Good Clinical Practice ICH. Available: . Accessed 2014 July 11.
    1. Lucchi NW, Demas A, Narayanan J, Sumari D, Kabanywanyi A, et al. (2010) Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria. PloS one 5: e13733.
    1. Patel JC, Lucchi NW, Srivastava P, Lin JT, Sug-Aram R, et al. (2014) Field Evaluation of a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Assay, RealAmp, for the Diagnosis of Malaria in Thailand and India. The Journal of infectious diseases
    1. Abdul-Ghani R, Al-Mekhlafi AM, Karanis P (2012) Loop-mediated isothermal amplification (LAMP) for malarial parasites of humans: would it come to clinical reality as a point-of-care test? Acta tropica 122: 233–240.

Source: PubMed

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