Abstract
Background
Prompt malarial treatment and surveillance is crucial for accurate diagnosis of Plasmodium Sp. Gold standard microscopic examination has been widely applied for diagnosis of malaria in most part of the endemic areas. But in case of submicroscopic and asymptomatic microscopic diagnosis is questioned. The study aims to develop a simple, cost effective & robust nucleic acid amplification technique for the detection of malaria parasite.
Methods
Study population included 50 clinically diagnosed positive malaria patient samples from various pathological laboratories. Microscopy by preparing thick film was carried out of every sample for primary screening in the available facility of Surat Raktadan Kendra & Research Centre- Blood Bank. The conventional PCR (Polymerase Chain Reaction) was applied for genus-specific amplification targeting the 18 S rRNA gene of Plasmodium. Agarose gel electrophoresis was used to separate and analyze the amplified PCR product using 2% Agarose gel.
Results and Conclusion
The study shows that nested PCR not only detected all microscopic positive samples, but also detected submicroscopic infections that were missed or misread by microscopy. Hence, the sensitivity of molecular based detection technique is proved to be more compared to microscopic examination.
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References
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 77:119–127. https://doi.org/10.4269/ajtmh.2007.77.119
Fançony C, Sebastiao YV, Pires JE, Gamboa D, Nery SV (2013) Performance of microscopy and RDTs in the context of a malaria prevalence survey in Angola: a comparison using PCR as the gold standard. Malar J 12:284. https://doi.org/10.1186/1475-2875-12-284
Global Malaria Programme (GMP). World malaria report (2008) Geneva: WHO/HTM/GMP/2008.1. ISBN: 978 92 4 156369 7
Payne D (1988) Use and limitation of light microscopy for diagnosing malaria at the primary health care level. Bull World Health Organ 66:621–626
Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown KN (1993) Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol Biochem Parasitol 58:283–292. https://doi.org/10.1016/0166-6851(93)90050-8
Snounou G, Singh B (2002) Nested PCR analysis of Plasmodium parasites. Methods Mol Med 72:189–203. https://doi.org/10.1385/1-59259-271-6:189
Roper C, Elhassan IM, Hviid L, Giha H, Richardson et al (1996) Detection of very low level Plasmodium Falciparum infections using the nested polymerase chain reaction and a reassessment of the epidemiology of unstable malaria in Sudan. Am J Trop Med Hyg 54:325–331. https://doi.org/10.4269/ajtmh.1996.54.325
Kimura M, Kaneko O, Liu Q, Zhou M, Kawamoto F, Wataya Y et al (1997) Identification of the four species of human malaria parasites by nested PCR that targets variant sequences in the small subunit rRNA gene. Parasitol Int 46:91–95. https://doi.org/10.1016/s1383-5769(97)00013-5
Perandin F, Manca N, Calderaro A, Piccolo G, Galati L, Ricci L 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 42:1214–1219. https://doi.org/10.1128/jcm.42.3.1214-1219.2004
Ebrahimzadeh A, Fouladi B, Fazaeli A (2007) High rate of detection of mixed infections of Plasmodium Vivax and Plasmodium Falciparum in South–East of Iran, using nested PCR. Parasitol Int 56:61–64. https://doi.org/10.1016/j.parint.2006.12.001
Krishna S, Bharti PK, Chandel HS, Ahmad A, Kumar R, Singh PP et al (2015) Detection of mixed infections with Plasmodium spp. by PCR, India, 2014. Emerg Infect Dis 21:1853–1857. https://doi.org/10.3201/eid2110.150678
Mekonnen SK, Aseffa A, Medhin G, Berhe N, Velavan TP (2014) Re-evaluation of microscopy confirmed Plasmodium Falciparum and Plasmodium Vivax malaria by nested PCR detection in southern Ethiopia. Malar J 13:48. https://doi.org/10.1186/1475-2875-13-48
Sitali L, Chipeta J, Miller JM, Moonga HB, Kumar N, Moss WJ et al (2015) Patterns of mixed Plasmodium species infections among children six years and under in selected malaria hyper–endemic communities of Zambia: population–based survey observations. BMC Infect Dis 15:204. https://doi.org/10.1186/s12879-015-0935-7
Kumar A, Valecha N, Jain T, Dash AP (2007) Burden of malaria in India: retrospective and prospective view. Am J Trop Med Hyg 77:69–78. https://doi.org/10.4269/ajtmh.2007.77.69
Patil RR, Kumar RK (2011) World bank EMCP malaria project in Orissa, India - A field reality. Trop Parasitol 1(1):26–29. https://doi.org/10.4103/2229-5070.72111
The Indian Express (2019) Telling Numbers: Malaria drop is sharpest in India, 90% of cases are in 7 states. https://indianexpress.com/article/explained/malaria-drop-is-sharpest-in-india-90-of-cases-are-in-7-states-6151060/
Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosario VE, Thaithong S, Brown KN (1993) High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol 61:315–320. https://doi.org/10.1016/0166-6851(93)90077-b
Shah A, Patel P, Jariwala K, Qureshi F, Mishra K, Bharadva S, Ghosh K (2022) Molecular genotyping of Indian blood group antigens amongst regular voluntary blood donors of Surat city, Gujarat, India. Transfus Apher Sci 61:103325. https://doi.org/10.1016/j.transci.2021.103325
Johnston SP, Pieniazek NJ, Xayavong MV, Slemenda SB, Wilkins PP, da Silva AJ (2006) PCR as a confirmatory technique for laboratory diagnosis of malaria. J Clin Microbiol 44:1087–1089. https://doi.org/10.1128/jcm.44.3.1087-1089.2006
de Roode JC, Culleton R, Bell AS, Read AF (2004) Competitive release of drug resistance following drug treatment of mixed Plasmodium chabaudi infections. Malar J 3:33. https://doi.org/10.1186/1475-2875-3-33
Mayxay M, Pukrittayakamee S, Newton PN, White NJ (2004) Mixed-species malaria infections in humans. Trends Parasitol 20:233–240. https://doi.org/10.1016/j.pt.2004.03.006
Lee G, Jeon E, Le Tien D, Kim T, Yoo J, Yong Kim H et al (2011) Development and evaluation of a rapid diagnostic test for P. Falciparum, P. Vivax and mixed-species malaria antigens. Am J Trop Med Hyg 85:989–993. https://doi.org/10.4269/ajtmh.2011.11-0265
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Patel, P., Mishra, K.K. & Ghosh, K. Confirmatory Assay for Laboratory Diagnosis of Malaria Using Molecular Approach. Acta Parasit. 69, 1000–1004 (2024). https://doi.org/10.1007/s11686-024-00831-0
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DOI: https://doi.org/10.1007/s11686-024-00831-0