• 1.

    WHO , 2020. World Malaria Report 2020: 20 Years of Global Progress and Challenges. Geneva, Switzerland: World Health Organization.

  • 2.

    Ochola LB, Vounatsou P, Smith T, Mabaso MLH, Newton C, 2006. The reliability of diagnostic techniques in the diagnosis and management of malaria in the absence of a gold standard. Lancet Infect Dis 6: 582588.

    • Search Google Scholar
    • Export Citation
  • 3.

    Ndao M, Bandyayera E, Kokoskin E, Gyorkos TW, MacLean JD, Ward BJ, 2004. Comparison of blood smear, antigen detection, and nested-PCR methods for screening refugees from regions where malaria is endemic after a malaria outbreak in Quebec, Canada. J Clin Microbiol 42: 26942700.

    • Search Google Scholar
    • Export Citation
  • 4.

    Gamboa D et al., 2010. A large proportion of P. falciparum isolates in the Amazon region of Peru lack pfhrp2 and pfhrp3: implications for malaria rapid diagnostic tests. PLoS One 5: e8091.

    • Search Google Scholar
    • Export Citation
  • 5.

    Parr JB et al., 2017. Pfhrp2-deleted Plasmodium falciparum parasites in the Democratic Republic of Congo: a national cross-sectional survey. J Infect Dis 216: 33–44.

    • Search Google Scholar
    • Export Citation
  • 6.

    Fradejas I et al., 2019. Prevalence of submicroscopic malaria infection in immigrants living in Spain. Malar J 18: 242.

  • 7.

    Bousema JT, Gouagna LC, Drakeley CJ, Meutstege AM, Okech BA, Akim IN, Beier JC, Githure JI, Sauerwein RW, 2004. Plasmodium falciparum gametocyte carriage in asymptomatic children in western Kenya. Malar J 3: 18.

    • Search Google Scholar
    • Export Citation
  • 8.

    Alves FP, Gil LHS, Marrelli MT, Ribolla PEM, Camargo EP, 2005. Asymptomatic carriers of Plasmodium spp. as infection source for malaria vector mosquitoes in the Brazilian Amazon. J Med Entomol 42: 777779.

    • Search Google Scholar
    • Export Citation
  • 9.

    Jiram AI, Ooi CH, Rubio JM, Hisam S, Karnan G, Sukor NM, Artic MM, Ismail NP, Alias NW, 2019. Evidence of asymptomatic submicroscopic malaria in low transmission areas in Belaga district, Kapit division, Sarawak, Malaysia. Malar J 18: 156.

    • Search Google Scholar
    • Export Citation
  • 10.

    Das S et al., 2017. Performance of a high-sensitivity rapid diagnostic test for Plasmodium falciparum malaria in asymptomatic individuals from Uganda and Myanmar and naive human challenge infections. Am J Trop Med Hyg 97: 15401550.

    • Search Google Scholar
    • Export Citation
  • 11.

    Berzosa P et al., 2018. Comparison of three diagnostic methods (microscopy, RDT, and PCR) for the detection of malaria parasites in representative samples from Equatorial Guinea. Malar J 17: 333.

    • Search Google Scholar
    • Export Citation
  • 12.

    Hagen RM, Hinz R, Tannich E, Frickmann H, 2015. Comparison of two real-time PCR assays for the detection of malaria parasites from hemolytic blood samples—short communication. Eur J Microbiol Immunol (Bp) 5: 159163.

    • Search Google Scholar
    • Export Citation
  • 13.

    Torrús D, Carranza C, Manuel Ramos J, Carlos Rodríguez J, Rubio JM, Subirats M, Ta-Tang T-H, 2015. Diagnóstico microbiológico de la malaria importada. Enferm Infecc Microbiol Clin 33: 4046.

    • Search Google Scholar
    • Export Citation
  • 14.

    Singh US, Siwal N, Pande V, Das A, 2017. Can mixed parasite infections thwart targeted malaria elimination program in India? BioMed Res Int 2017: 111.

    • Search Google Scholar
    • Export Citation
  • 15.

    Mayxay M, Pukrittayakamee S, Newton PN, White NJ, 2004. Mixed-species malaria infections in humans. Trends Parasitol 20: 233240.

  • 16.

    Kim G, Hong H-L, Kim SY, Lee HR, Kim DG, Park S, Shin H-S, Chin BS, Kim Y, 2019. Mixed Infection with Plasmodium falciparum and Plasmodium ovale in a returned traveller: the first case in Korea. J Korean Med Sci 34: e23.

    • Search Google Scholar
    • Export Citation
  • 17.

    Kotepui M, Kotepui KU, De Jesus Milanez G, Masangkay FR, 2020. Plasmodium spp. mixed infection leading to severe malaria: a systematic review and meta-analysis. Sci Rep 10: 11068.

    • Search Google Scholar
    • Export Citation
  • 18.

    Singh B, Bobogare A, Cox-Singh J, Snounou G, Abdullah MS, Rahman HA, 1999. A genus-and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am J Trop Med Hyg 60: 687692.

    • Search Google Scholar
    • Export Citation
  • 19.

    Rubio J, Post R, van Leeuwen W, Henry M-C, Lindergard G, Hommel M, 2002. Alternative polymerase chain reaction method to identify Plasmodium species in human blood samples: the semi-nested multiplex malaria PCR (SnM-PCR). Trans R Soc Trop Med Hyg 96 (Suppl_1 ):S199S204.

    • Search Google Scholar
    • Export Citation
  • 20.

    Perandin F et al., 2004. Development of a Real-Time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis. J Clin Microbiol 42: 12141219.

    • Search Google Scholar
    • Export Citation
  • 21.

    Roth JM, Korevaar DA, Leeflang MMG, Mens PF, 2016. Molecular malaria diagnostics: a systematic review and meta-analysis. Crit Rev Clin Lab Sci 53: 87105.

    • Search Google Scholar
    • Export Citation
  • 22.

    Phillips MA, Burrows JN, Manyando C, van Huijsduijnen RH, Van Voorhis WC, Wells TNC, 2017. Malaria. Nat Rev Dis Primers 3: 17050.

  • 23.

    Notomi T, 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28: E63.

  • 24.

    Tomita N, Mori Y, Kanda H, Notomi T, 2008. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3: 877882.

    • Search Google Scholar
    • Export Citation
  • 25.

    Han E-T, 2013. Loop-mediated isothermal amplification test for the molecular diagnosis of malaria. Expert Rev Mol Diagn 13: 205218.

  • 26.

    Gachugia J, Chebore W, Otieno K, Ngugi CW, Godana A, Kariuki S, 2020. Evaluation of the colorimetric malachite green loop-mediated isothermal amplification (MG-LAMP) assay for the detection of malaria species at two different health facilities in a malaria endemic area of western Kenya. Malar J 19: 329.

    • Search Google Scholar
    • Export Citation
  • 27.

    Kudyba HM, Louzada J, Ljolje D, Kudyba KA, Muralidharan V, Oliveira-Ferreira J, Lucchi NW, 2019. Field evaluation of malaria malachite green loop-mediated isothermal amplification in health posts in Roraima state, Brazil. Malar J 18: 98.

    • Search Google Scholar
    • Export Citation
  • 28.

    Ocker R, Prompunjai Y, Chutipongvivate S, Karanis P, 2016. Malaria diagnosis by loop-mediated isothermal amplification (LAMP) in Thailand. Rev Inst Med Trop São Paulo 58: 27.

    • Search Google Scholar
    • Export Citation
  • 29.

    Lucchi NW, Ndiaye D, Britton S, Udhayakumar V, 2018. Expanding the malaria molecular diagnostic options: opportunities and challenges for loop-mediated isothermal amplification tests for malaria control and elimination. Expert Rev Mol Diagn 18: 195203.

    • Search Google Scholar
    • Export Citation
  • 30.

    Ljolje D, Abdallah R, Lucchi NW, 2021. Detection of malaria parasites in samples from returning US travelers using the Alethia® Malaria Plus LAMP assay. BMC Res Notes 14: 128.

    • Search Google Scholar
    • Export Citation
  • 31.

    Cuadros J, Martin Ramírez A, González IJ, Ding XC, Perez Tanoira R, Rojo-Marcos G, Gómez-Herruz P, Rubio JM, 2017. LAMP kit for diagnosis of non-falciparum malaria in Plasmodium ovale infected patients. Malar J 16: 20.

    • Search Google Scholar
    • Export Citation
  • 32.

    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. J Clin Microbiol 47: 975980.

    • Search Google Scholar
    • Export Citation
  • 33.

    Altona Diagnostics , 2018. Instructions for Use. RealStar® Malaria Screen & Type.

  • 34.

    Miguel-Oteo M, Jiram AI, Ta-Tang TH, Lanza M, Hisam S, Rubio JM, 2017. Nested multiplex PCR for identification and detection of human Plasmodium species including Plasmodium knowlesi. Asian Pac J Trop Med 10: 299304.

    • Search Google Scholar
    • Export Citation
  • 35.

    Altona Diagnostics , 2019. RealStar® Malaria PCR Kit - Altona-Diagnostics EN. Available at: https://altona-diagnostics.com/en/products/reagents-140/reagents/realstar-real-time-pcr-reagents/realstar-malaria-pcr-kit-ce.html. Accessed May 20, 2019.

  • 36.

    Saah AJ, 1997. “Sensitivity” and “specificity” reconsidered: the meaning of these terms in analytical and diagnostic settings. Ann Intern Med 126: 91.

    • Search Google Scholar
    • Export Citation
  • 37.

    Santiago Perez M, Hervada Vidal X, Naveira Barbeito G, Silva L, Fariñas H, Vazquez E, Bacallao J, Mújica O, 2010. The epidat programme: uses and perspectives. Rev Panam Salud Pública27: 80–82.

  • 38.

    Frickmann H, Wegner C, Ruben S, Behrens C, Kollenda H, Hinz R, Rojak S, Schwarz NG, Hagen RM, Tannich E, 2019. Evaluation of the multiplex real-time PCR assays RealStar malaria S&T PCR kit 1.0 and FTD malaria differentiation for the differentiation of Plasmodium species in clinical samples. Travel Med Infect Dis 31: 101442.

    • Search Google Scholar
    • Export Citation
  • 39.

    Singh B, Sung LK, Matusop A, Radhakrishnan A, Shamsul SS, Cox-Singh J, Thomas A, Conway DJ, 2004. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet 363: 10171024.

    • Search Google Scholar
    • Export Citation
  • 40.

    Müller M, Schlagenhauf P, 2014. Plasmodium knowlesi in travellers, update 2014. Int J Infect Dis 22: 5564.

  • 41.

    Ta Tang T-H, Salas A, Ali-Tammam M, Martinez M, del C, Lanza M, Arroyo E, Rubio JM., 2010. First case of detection of Plasmodium knowlesi in Spain by real time PCR in a traveller from Southeast Asia. Malar J 9: 219.

    • Search Google Scholar
    • Export Citation
  • 42.

    Rougemont M, Van Saanen M, Sahli R, Hinrikson HP, Bille J, Jaton K, 2004. Detection of Four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays. J Clin Microbiol 42: 56365643.

    • Search Google Scholar
    • Export Citation
  • 43.

    Bialasiewicz S, Whiley DM, Nissen MD, Sloots TP, 2007. Impact of Competitive Inhibition and Sequence Variation upon the Sensitivity of Malaria PCR. J Clin Microbiol45: 1621–1623.

 
 
 

 

 

 

 

 

 

Assessment of Commercial Real-Time PCR Assays for Detection of Malaria Infection in a Non-Endemic Setting

View More View Less
  • 1 Malaria and Parasitic Emerging Diseases Laboratory, National Microbiology Center, Instituto de Salud Carlos III, Madrid, Spain;
  • | 2 NTD Laboratory, Tropical Medicine Center, Instituto de Salud Carlos III, Madrid, Spain;
  • | 3 Parasitology Unit, Infectious Disease Research Centre, Institute for Medical Research, National Institute of Health, Setia Alam, Selangor, Malaysia

ABSTRACT.

Malaria control and elimination require prompt diagnosis and accurate treatment. Conventional methods such as rapid diagnostic tests (RDTs) and microscopy lack the characteristics to detect low parasitemias, commonly found in asymptomatic parasitemias and/or submicroscopic malaria carriers. On the contrary, molecular methods have higher sensitivity and specificity. This study evaluated the performance of two commercial real-time polymerase chain reaction (PCR) assays, RealStar® Malaria PCR (RealStar-genus) and RealStar Malaria Screen&Type PCR (RealStar-species), compared with the reference Nested Multiplex Malaria PCR, for the detection of the main five Plasmodium species affecting humans. A total of 121 samples were evaluated. Values of sensitivity (98.9% and 97.8%) and specificity (100% and 96.7%) of the RealStar-genus and the RealStar-species assays, respectively, were very good. The limit of detection (LoD) for the RealStar-genus assay showed a mean value of 0.28 parasites/µL with Plasmodium falciparum samples; while, the LoD of the RealStar-species assay ranged from 0.09 parasites/µL for P. vivax to two parasites/µL for P. ovale. The time to complete a diagnosis was established in 4 hours. Our findings showed a very good concordance of both assays compared with the reference method, with a very good analytical sensitivity. RealStar-species assay was able to correctly characterize double and triple infections. Therefore, these RealStar assays have shown to be useful tools in malaria diagnosis in non-endemic countries and even endemic countries, and for malaria control in general, detecting low parasitemias with sensitivity similar to the most sensitive methods as nested PCR, but with lower time to get the results.

Author Notes

Address correspondence to José M. Rubio, Malaria and Parasitic Emerging Diseases Laboratory, National Microbiology Center, Instituto de Salud Carlos III, Madrid, Spain. E-mail: jmrubio@isciii.es

Financial support: This work was funded by projects PI14CIII/00014 and PI17CIII/00035 from the Instituto de Salud Carlos III (Ministry of Science and Innovation) and cofounded by the European Regional Development Fund. Altona Diagnostics also partially funded this study by donating the kits and funding the necessary consumables. Alexandra Martin Ramirez is supported by an ISCIII Rio Hortega contract.

Disclaimer: The funders, including Altona Diagnostic, have not participated in the collection, analysis, and interpretation of data, in the writing of the report, and in the decision to submit the article for publication.

Authors’ Addresses: Alexandra Martín Ramírez, Marta Lanza Suárez, Ana Álvarez Fernández, Carlota Muñoz García, and José M. Rubio, Malaria and Parasitic Emerging Diseases Laboratory, National Microbiology Center, Instituto de Salud Carlos III, Madrid, Spain, E-mails: a.martin@isciii.es, mlanza@isciii.es, anamaf_g@hotmail.com, carlota.mg114@gmail.com, and jmrubio@isciii.es. Thuy Huong Ta Tang, Malaria and Parasitic Emerging Diseases Laboratory, National Microbiology Center, Instituto de Salud Carlos III, Madrid, Spain, and NTD Laboratory, Tropical Medicine Center, Instituto de Salud Carlos III, Madrid, Spain, E-mail: tta@isciii.es. Shamilah Hisam, Parasitology Unit, Infectious Disease Research Centre, Institute for Medical Research, National Institute of Health, Setia Alam, Selangor, Malaysia, E-mail: shamilah@imr.gov.my.

Save