Comparison of diagnostic methods for the detection and quantification of the four sympatric Plasmodium species in field samples from Papua New Guinea

Anna Rosanas-Urgell, Dania Mueller, Inoni Betuela, Céline Barnadas, Jonah Iga, Peter A Zimmerman, Hernando A del Portillo, Peter Siba, Ivo Mueller, Ingrid Felger, Anna Rosanas-Urgell, Dania Mueller, Inoni Betuela, Céline Barnadas, Jonah Iga, Peter A Zimmerman, Hernando A del Portillo, Peter Siba, Ivo Mueller, Ingrid Felger

Abstract

Background: Accurate diagnosis of Plasmodium infections is essential for malaria morbidity and mortality reduction in tropical areas. Despite great advantages of light microscopy (LM) for malaria diagnosis, its limited sensitivity is a critical shortfall for epidemiological studies. Robust molecular diagnostics tools are thus needed.

Methods: The present study describes the development of a duplex quantitative real time PCR (qPCR) assay, which specifically detects and quantifies the four human Plasmodium species. Performance of this method was compared to PCR-ligase detection reaction-fluorescent microsphere assay (PCR_LDR_FMA), nested PCR (nPCR) and LM, using field samples collected from 452 children one to five years of age from the Sepik area in Papua New Guinea. Agreement between diagnostic methods was calcualted using kappa statistics.

Results: The agreement of qPCR with other molecular diagnostic methods was substantial for the detection of P. falciparum, but was moderate for the detection of P. vivax, P. malariae and P. ovale. P. falciparum and P. vivax prevalence by qPCR was 40.9% and 65.7% respectively. This compares to 43.8% and 73.2% by nPCR and 47.1% and 67.5% by PCR_LDR_FMA. P. malariae and P. ovale prevalence was 4.7% and 7.3% by qPCR, 3.3% and 3.8% by nPCR, and 7.7% and 4.4% by PCR_LDR_FMA. Prevalence by LM was lower for all four species, being 25.4% for P. falciparum, 54.9% for P. vivax, 2.4% for P. malariae and 0.0% for P. ovale. The quantification by qPCR closely correlated with microscopic quantification for P. falciparum and P. vivax samples (R2 = 0.825 and R2 = 0.505, respectively). The low prevalence of P. malariae and P. ovale did not permit a solid comparative analysis of quantification for these species.

Conclusions: The qPCR assay developed proved optimal for detection of all four Plasmodium species. Densities by LM were well reflected in quantification results by qPCR, whereby congruence was better for P. falciparum than for P. vivax. This likely is a consequence of the generally lower P. vivax densities. Easy performance of the qPCR assay, a less laborious workflow and reduced risk of contamination, together with reduced costs per sample through reduced reaction volume, opens the possibility to implement qPCR in endemic settings as a suitable diagnostic tool for large epidemiological studies.

Figures

Figure 1
Figure 1
Comparison of quantification assessed by light microscopy versus qPCR.

References

    1. Mehlotra RK, Kasehagen LJ, Baisor M, Lorry K, Kazura JW, Bockarie MJ, Zimmerman PA. Malaria infections are randomly distributed in diverse holoendemic areas of Papua New Guinea. Am J Trop Med Hyg. 2002;67:555–562.
    1. Mueller I, Galinski MR, Baird JK, Carlton JM, Kochar DK, Alonso PL, del Portillo HA. Key gaps in the knowledge of Plasmodium vivax, a neglected human malaria parasite. Lancet Infect Dis. 2009;9:555–566. doi: 10.1016/S1473-3099(09)70177-X.
    1. Organization WH. World malaria report 2008. Geneva, Switzerland: WHO Press; 2008.
    1. Moody AH, Chiodini PL. Non-microscopic method for malaria diagnosis using OptiMAL IT, a second-generation dipstick for malaria pLDH antigen detection. Br J Biomed Sci. 2002;59:228–231.
    1. Coleman RE, Sattabongkot J, Promstaporm S, Maneechai N, Tippayachai B, Kengluecha A, Rachapaew N, Zollner G, Miller RS, Vaughan JA, Thimasarn K, Khuntirat B. Comparison of PCR and microscopy for the detection of asymptomatic malaria in a Plasmodium falciparum/vivax endemic area in Thailand. Malar J. 2006;5:121. doi: 10.1186/1475-2875-5-121.
    1. Ochola LB, Vounatsou P, Smith T, Mabaso ML, Newton CR. The reliability of diagnostic techniques in the diagnosis and management of malaria in the absence of a gold standard. Lancet Infect Dis. 2006;6:582–588. doi: 10.1016/S1473-3099(06)70579-5.
    1. WHO. Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: round 1 (2008) Geneva: World Health Organization; 2009.
    1. de Monbrison F, Angei C, Staal A, Kaiser K, Picot S. Simultaneous identification of the four human Plasmodium species and quantification of Plasmodium DNA load in human blood by real-time polymerase chain reaction. Trans R Soc Trop Med Hyg. 2003;97:387–390. doi: 10.1016/S0035-9203(03)90065-4.
    1. Rubio JM, Benito A, Roche J, Berzosa PJ, Garcia ML, Mico M, Edu M, Alvar J. Semi-nested, multiplex polymerase chain reaction for detection of human malaria parasites and evidence of Plasmodium vivax infection in Equatorial Guinea. Am J Trop Med Hyg. 1999;60:183–187.
    1. Lee MA, Tan CH, Aw LT, Tang CS, Singh M, Lee SH, Chia HP, Yap EP. Real-time fluorescence-based PCR for detection of malaria parasites. J Clin Microbiol. 2002;40:4343–4345. doi: 10.1128/JCM.40.11.4343-4345.2002.
    1. Farcas GA, Zhong KJ, Mazzulli T, Kain KC. Evaluation of the RealArt Malaria LC real-time PCR assay for malaria diagnosis. J Clin Microbiol. 2004;42:636–638. doi: 10.1128/JCM.42.2.636-638.2004.
    1. Mangold KA, Manson RU, Koay ES, Stephens L, Regner M, Thomson RB Jr, Peterson LR, Kaul KL. Real-time PCR for detection and identification of Plasmodium spp. J Clin Microbiol. 2005;43:2435–2440. doi: 10.1128/JCM.43.5.2435-2440.2005.
    1. Rougemont M, Van Saanen M, Sahli R, Hinrikson HP, Bille J, Jaton K. Detection of four Plasmodium species in blood from humans by 18 S rRNA gene subunit-based and species-specific real-time PCR assays. J Clin Microbiol. 2004;42:5636–5643. doi: 10.1128/JCM.42.12.5636-5643.2004.
    1. Bialasiewicz S, Whiley DM, Nissen MD, Sloots TP. Impact of competitive inhibition and sequence variation upon the sensitivity of malaria PCR. J Clin Microbiol. 2007;45:1621–1623. doi: 10.1128/JCM.02145-06.
    1. Perandin F, Manca N, Calderaro A, Piccolo G, Galati L, Ricci L, Medici MC, Arcangeletti MC, Snounou G, Dettori G. et al.Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis. J Clin Microbiol. 2004;42:1214–1219. doi: 10.1128/JCM.42.3.1214-1219.2004.
    1. 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;47:975–980. doi: 10.1128/JCM.01858-08.
    1. Dormond L, Jaton-Ogay K, de Valliere S, Genton B, Bille J, Greub G. Multiplex real-time PCR for the diagnosis of malaria: correlation with microscopy. Clin Microbiol Infect. 2010. in press .
    1. Vo TK, Bigot P, Gazin P, Sinou V, De Pina JJ, Huynh DC, Fumoux F, Parzy D. Evaluation of a real-time PCR assay for malaria diagnosis in patients from Vietnam and in returned travellers. Trans R Soc Trop Med Hyg. 2007;101:422–428. doi: 10.1016/j.trstmh.2006.09.004.
    1. McNamara DT, Kasehagen LJ, Grimberg BT, Cole-Tobian J, Collins WE, Zimmerman PA. Diagnosing infection levels of four human malaria parasite species by a polymerase chain reaction/ligase detection reaction fluorescent microsphere-based assay. Am J Trop Med Hyg. 2006;74:413–421.
    1. Genton B, al-Yaman F, Beck HP, Hii J, Mellor S, Rare L, Ginny M, Smith T, Alpers MP. The epidemiology of malaria in the Wosera area, East Sepik Province, Papua New Guinea, in preparation for vaccine trials. II. Mortality and morbidity. Ann Trop Med Parasitol. 1995;89:377–390.
    1. al-Yaman F, Genton B, Kramer KJ, Taraika J, Chang SP, Hui GS, Alpers MP. Acquired antibody levels to Plasmodium falciparum merozoite surface antigen 1 in residents of a highly endemic area of Papua New Guinea. Trans R Soc Trop Med Hyg. 1995;89:555–559. doi: 10.1016/0035-9203(95)90106-X.
    1. Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown KN. 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. 1993;58:283–292. doi: 10.1016/0166-6851(93)90050-8.
    1. Bustin SA. Why the need for qPCR publication guidelines?--The case for MIQE. Methods. pp. 217–226.
    1. Landis JR, Koch GG. An application of hierarchical kappa-type statistics in the assessment of majority agreement among multiple observers. Biometrics. 1977;33:363–374. doi: 10.2307/2529786.
    1. Mens P, Spieker N, Omar S, Heijnen M, Schallig H, Kager PA. Is molecular biology the best alternative for diagnosis of malaria to microscopy? A comparison between microscopy, antigen detection and molecular tests in rural Kenya and urban Tanzania. Trop Med Int Health. 2007;12:238–244.
    1. Mixson-Hayden T, Lucchi NW, Udhayakumar V. Evaluation of three PCR-based diagnostic assays for detecting mixed Plasmodium infection. BMC Res Notes. 2010;3:88. doi: 10.1186/1756-0500-3-88.
    1. Koukounari A, Webster JP, Donnelly CA, Bray BC, Naples J, Bosompem K, Shiff C. Sensitivities and specificities of diagnostic tests and infection prevalence of Schistosoma haematobium estimated from data on adults in villages northwest of Accra, Ghana. Am J Trop Med Hyg. 2009;80:435–441.
    1. de Clare Bronsvoort BM, von Wissmann B, Fevre EM, Handel IG, Picozzi K, Welburn SC. No gold standard estimation of the sensitivity and specificity of two molecular diagnostic protocols for Trypanosoma brucei spp. in Western Kenya. PLoS One. 2010;5:e8628. doi: 10.1371/journal.pone.0008628.
    1. Faucher JF, Aubouy A, Beheton T, Makoutode P, Abiou G, Doritchamou J, Houze P, Ouendo E, Deloron P, Cot M. What would PCR assessment change in the management of fevers in a malaria endemic area? A school-based study in Benin in children with and without fever. Malar J. 2010;9:224. doi: 10.1186/1475-2875-9-224.

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