Abstract
The conventional molecular diagnosis of malaria uses 18S rRNA-based PCR assay employing blood samples. This assay presents limitation in terms of long turnaround time and increased chances of false-positive results. Here, we evaluated one-step singleplex or multiplex PCR assay based on high copy species-specific consensus repeat sequences (CRS) along with standard 18S rRNA nested PCR (18S n-PCR) assay to detect P. falciparum and P. vivax infection using blood and saliva samples from Indian febrile patients. Out of 327 patients, 187 were found to be positive for malaria parasites by microscopic examination of peripheral blood smears. Among these 130 were P. vivax and 57 were P. falciparum cases. The18S n-PCR assay and CRS PCR assay identified 186 out of 187 cases (99.4 %). Multiplex CRS PCR assay detected Plasmodium in 176 out of 187 cases (94.1 %). Both singleplex and multiplex CRS PCR assay identified 6 mixed infection cases, while 18S n-PCR assay detected 10 mixed infection cases of P. vivax and P. falciparum, which were not recognized by microscopy. Non-invasive Plasmodium detection rate with DNA derived from saliva samples was highest for 18S n-PCR (87.36 %), followed by singleplex CRS (81 %) and multiplex CRS PCR assay (70.5 %). Specificity for P. vivax and P. falciparum detection for all assays was 98.48 % and 100 % respectively. Detection rate for P. vivax in saliva correlated with parasite density for CRS target-based assays. The species-specific CRS PCR, either as a singleplex or multiplex assay, can have an impact on diagnosis and epidemiological studies in malaria.
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References
WHO (2013) World Malaria Report 2013. World Health Organization, Geneva
Gupta B, Gupta P, Sharma A, Singh V, Dash AP, Das A (2010) High proportion of mixed-species Plasmodium infections in India revealed by PCR diagnostic assay. Trop Med Int Health 15(7):819–824
Baird JK (2013) Evidence and implications of mortality associated with acute Plasmodium vivax malaria. Clin Microbiol Rev 26(1):36–57
Mueller I, Galinski MR, Baird JK, Carlton JM, Kochar DK, Alonso PL, del Portillo HA (2009) Key gaps in the knowledge of Plasmodium vivax, a neglected human malaria parasite. Lancet Infect Dis 9(9):555–566
Valecha N, Pinto RG, Turner GD, Kumar A, Rodrigues S, Dubhashi NG, Rodrigues E, Banaulikar SS, Singh R, Dash AP, Baird JK (2009) Histopathology of fatal respiratory distress caused by Plasmodium vivax malaria. Am J Trop Med Hyg 81(5):758–762
Dorsey G, Gandhi M, Oyugi JH, Rosenthal PJ (2000) Difficulties in the prevention, diagnosis, and treatment of imported malaria. Arch Intern Med 160(16):2505–2510
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 (6 Suppl):119–127
McMorrow ML, Aidoo M, Kachur SP (2011) Malaria rapid diagnostic tests in elimination settings–can they find the last parasite? Clin Microbiol Infect 17(11):1624–1631
Kumar N, Pande V, Bhatt RM, Shah NK, Mishra N, Srivastava B, Valecha N, Anvikar AR (2013) Genetic deletion of HRP2 and HRP3 in Indian Plasmodium falciparum population and false negative malaria rapid diagnostic test. Acta Trop 125(1):119–121
Koita OA, Doumbo OK, Ouattara A, Tall LK, Konare A, Diakite M, Diallo M, Sagara I, Masinde GL, Doumbo SN, Dolo A, Tounkara A, Traore I, Krogstad DJ (2012) False-negative rapid diagnostic tests for malaria and deletion of the histidine-rich repeat region of the hrp2 gene. Am J Trop Med Hyg 86(2):194–198
Hanscheid T, Grobusch MP (2002) How useful is PCR in the diagnosis of malaria? Trends Parasitol 18(9):395–398
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(2):283–292
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(4):687–692
Singh B, Kim Sung L, 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(9414):1017–1024
Perandin F, Manca N, Calderaro A, Piccolo G, Galati L, Ricci L, Medici MC, Arcangeletti MC, Snounou G, Dettori G, Chezzi C (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(3):1214–1219
Han ET, Watanabe R, Sattabongkot J, Khuntirat B, Sirichaisinthop J, Iriko H, Jin L, Takeo S, Tsuboi T (2007) Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis. J Clin Microbiol 45(8):2521–2528
Demas A, Oberstaller J, DeBarry J, Lucchi NW, Srinivasamoorthy G, Sumari D, Kabanywanyi AM, Villegas L, Escalante AA, Kachur SP, Barnwell JW, Peterson DS, Udhayakumar V, Kissinger JC (2011) Applied genomics: data mining reveals species-specific malaria diagnostic targets more sensitive than 18S rRNA. J Clin Microbiol 49(7):2411–2418
Singh R, Savargaonkar D, Bhatt R, Valecha N (2013) Rapid detection of Plasmodium vivax in saliva and blood using loop mediated isothermal amplification (LAMP) assay. J Infect 67(3):245–247
Putaporntip C, Buppan P, Jongwutiwes S (2011) Improved performance with saliva and urine as alternative DNA sources for malaria diagnosis by mitochondrial DNA-based PCR assays. Clin Microbiol Infect 17(10):1484–1491
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 (2009) Towards high-throughput molecular detection of Plasmodium: new approaches and molecular markers. Malar J 8:86
Polley SD, Mori Y, Watson J, Perkins MD, Gonzalez IJ, Notomi T, Chiodini PL, Sutherland CJ (2010) Mitochondrial DNA targets increase sensitivity of malaria detection using loop-mediated isothermal amplification. J Clin Microbiol 48(8):2866–2871
Haanshuus CG, Mohn SC, Morch K, Langeland N, Blomberg B, Hanevik K (2013) A novel, single-amplification PCR targeting mitochondrial genome highly sensitive and specific in diagnosing malaria among returned travellers in Bergen, Norway. Malar J 12:26
Oyedeji SI, Awobode HO, Monday GC, Kendjo E, Kremsner PG, Kun JF (2007) Comparison of PCR-based detection of Plasmodium falciparum infections based on single and multicopy genes. Malar J 6:112
Filisetti D, Bombard S, N'Guiri C, Dahan R, Molet B, Abou-Bacar A, Hansmann Y, Christmann D, Candolfi E (2002) Prospective assessment of a new polymerase chain reaction target (STEVOR) for imported Plasmodium falciparum malaria. Eur J Clin Microbiol Infect Dis 21(9):679–681
Fontecha GA, Mendoza M, Banegas E, Poorak M, De Oliveira AM, Mancero T, Udhayakumar V, Lucchi NW, Mejia RE (2012) Comparison of molecular tests for the diagnosis of malaria in Honduras. Malar J 11:119
Yin J-H, Xia Z-G, Liu C-S, Zhou S-S (2013) Noninvasive sampling of saliva as an alternative way for malaria diagnosis: a systematic review. Sci J Microbiol. doi:10.7237/sjmb/180
Nwakanma DC, Gomez-Escobar N, Walther M, Crozier S, Dubovsky F, Malkin E, Locke E, Conway DJ (2009) Quantitative detection of Plasmodium falciparum DNA in saliva, blood, and urine. J Infect Dis 199(11):1567–1574
Buppan P, Putaporntip C, Pattanawong U, Seethamchai S, Jongwutiwes S (2010) Comparative detection of Plasmodium vivax and Plasmodium falciparum DNA in saliva and urine samples from symptomatic malaria patients in a low endemic area. Malar J 9:72
Kimura M, O K, Liu Q, Zhou M, Kawamoto F, Wataya Y, Otani S, Yamaguchi Y, Tanabe K (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(2):91–95
Boonma P, Christensen PR, Suwanarusk R, Price RN, Russell B, Lek-Uthai U (2007) Comparison of three molecular methods for the detection and speciation of Plasmodium vivax and Plasmodium falciparum. Malar J 6:124
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The study was supported by NIMR and a grant (5/8-7(231)2011-ECD-II) from the Indian Council of Medical Research, New Delhi, India. The technical support of Mrs Alka Kapoor is gratefully acknowledged.
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Singh, R., Singh, D.P., Gupta, R. et al. Comparison of three PCR-based assays for the non-invasive diagnosis of malaria: detection of Plasmodium parasites in blood and saliva. Eur J Clin Microbiol Infect Dis 33, 1631–1639 (2014). https://doi.org/10.1007/s10096-014-2121-z
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DOI: https://doi.org/10.1007/s10096-014-2121-z