Original Article

Simplified Pan-species Real-time PCR-based Detection of Plasmodium Spp. in Blood Smear

Abstract

Background: We aimed to quicken and simplify the detection of Plasmodium in blood samples by developing and testing a pan-Plasmodium real-time PCR for accurate screening of individuals suspected of malaria.

Methods: A single primer/probe set for pan-species Plasmodium-specific real time PCR targeting a conserved region of the small subunit 18S ribosomal DNA was designed and evaluated for rapid diagnosis and screening of malaria infections using dried blood smears. FTA cards were used for rapid and simple DNA extraction.

Results: The primers and probes showed a positive response with the DNA extracted from bloods infected with P. falciparum and P. vivax but not with DNA extracted from various smears from uninfected blood samples. Seven positive cases positive by both microscopy and nested PCR were found among 280 blood samples taken from in South and Southeast Iran. Five samples were identified as positive for P. vivax and two as positive for P. falciparum. All positive samples were positive by real-time PCR. Furthermore, all 38-blood samples positive by microscopy were positive by real-time PCR. No microscopy-negative samples were positive by real-time PCR.

Conclusion: By using a simple FTA card for DNA extraction and by application of the real-time PCR developed in this study, sensitivity similar to nested-PCR and microscopy was achieved. This format simplifies the detection of Plasmodium in large numbers of samples.

Caraballo H, King K. Emergency department management of mosquito-borne illness: malaria, dengue, and West Nile virus. Emerg Med Pract. 2014;16(5):1-23.

World Malaria Report 2015. Available from: http://www.who.int/malaria/publications/world-malaria-report-2015/report/en/

Worrall E, Basu S, Hanson K. Is malaria a disease of poverty? A review of the literature. Trop Med Int Health. 2005; 10(10):1047-59.

Gollin D, Zimmermann C. Malaria: Disease impacts and long-run income differences. 2007. Economics Working Papers. Paper 200730. http://digitalcommons.uconn.edu/econ_wpapers/200730

Hanafi-Bojd AA, Azari-Hamidian S, Vatandoost H, Charrahy Z. Spatio—temporal distribution of malaria vectors (Diptera: Culicidae) across different climatic zones of Iran. Asian Pac J Trop Med. 2011;4(6):498-504.

Zakeri S, Najafabadi ST, Zare A, Djadid ND. Detection of malaria parasites by nested PCR in south-eastern, Iran: evidence of highly mixed infections in Chahbahar district. Malaria J. 2002;1(1):2.

Turki H, Raeisi A, Malekzadeh K, Ghanbarnejad A, Zoghi S, Yeryan M, et al. Efficiency of Nested-PCR in detecting asymptomatic cases toward malaria elimination program in an endemic area of Iran. Iran J Parasitol. 2015;10(1):39-45.

Erdman LK, Kain KC. Molecular diagnostic and surveillance tools for global malaria control. Travel Med Infect Dis. 2008;6(1):82-99.

Taylor SM, Juliano JJ, Trottman PA, Griffin JB, Landis SH, Kitsa P, et al. High-throughput pooling and real-time PCR-based strategy for malaria detection. J Clin Microbiol. 2010;48(2):512-9.

Wang B, Han S-S, Cho C, Han J-H, Cheng Y, Lee S-K, et al. Comparison of microscopy, nested-PCR, and Real-Time-PCR assays using high-throughput screening of pooled samples for diagnosis of malaria in asymptomatic carriers from areas of endemicity in Myanmar. J Clin Microbiol. 2014;52(6):1838-45.

Lima GF, Levi JE, Geraldi MP, Sanchez MCA, Segurado AA, Hristov AD, et al. Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale. Mem Inst Oswaldo Cruz. 2011;106(6):691-700.

Hawkes M, Kain KC. Advances in malaria diagnosis. Expert Rev Anti Infect Ther. 2007; 5(3): 485-95.

Shokoples SE, Ndao M, Kowalewska-Grochowska K, Yanow SK. Multiplexed real-time PCR assay for discrimination of Plasmodium species with improved sensitivity for mixed infections. J Clin Microbiol. 2009; 47(4):975-80.

Snounou G, Pinheiro L, Gonçalves A, Fonseca L, Dias F, Brown KN, et al. The importance of sensitive detection of malaria parasites in the human and insect hosts in epidemiological studies, as shown by the analysis of field samples from Guinea Bissau. Trans Roy Soc Trop Med Hyg. 1993;87(6):649-53.

World Malaria Report 2008. http://www.who.int/malaria/publications/atoz/9789241563697/en/

Amexo M, Tolhurst R, Barnish G, Bates I. Malaria misdiagnosis: effects on the poor and vulnerable. Lancet. 2004;364(9448):1896-8.

Kain KC, MacPherson DW, Kelton T, Keystone JS, Mendelson J, MacLean JD. Malaria deaths in visitors to Canada and in Canadian travellers: a case series. CMAJ. 2001;164(5):654-9.

Martens P, Hall L. Malaria on the move: human population movement and malaria transmission. Emerg Infect Dis. 2000; 6(2):103-9.

Kain KC, Harrington MA, Tennyson S, Keystone JS. Imported malaria: prospective analysis of problems in diagnosis and management. Clin Infect Dis. 1998; 27(1):142-9.

Yamamura M, Makimura K, Ota Y. Evaluation of a new rapid molecular diagnostic system for Plasmodium falciparum combined with DNA filter paper, loop-mediated isothermal amplification, and melting curve analysis. Jpn J Infect Dis. 2009;62(1):20-5.

Files
IssueVol 11 No 4 (2016) QRcode
SectionOriginal Article(s)
Keywords
Malaria Plasmodium Diagnosis FTA cards Real-time PCR
Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
1.
HASSANPOUR G, MIRHENDI H, MOHEBALI M, RAEISI A, ZERAATI H, KESHAVARZ H. Simplified Pan-species Real-time PCR-based Detection of Plasmodium Spp. in Blood Smear. Iran J Parasitol. 1;11(4):463-470.