A direct comparison of real time PCR on plasma and blood to detect Plasmodium falciparum infection in children

Abigail A Lamikanra,Pau Cisteró,Llorenç Quinto,Alfons Jiménez,Augusto Nhabomba,Carlota Dobaño,Pedro L Alonso,Maria N Manaca,Hoi P Tsang,Alfredo Mayor,Ruth Aguilar,David J Roberts,Caterina Guinovart

doi:10.1186/1475-2875-11-201

Abstract

BackgroundEstimation of Plasmodium falciparum parasitaemia can vary with the method used and time of sampling. Quantitative real time PCR (qPCR) on whole blood or plasma samples has previously been shown to be more sensitive than thick film microscopy. However the efficiencies of each method have not been compared using samples obtained from infants less than one year old.MethodsA multiple of statistical approaches were used to compare the performance of qPCR on whole blood or plasma to detect the 18 S ribosomal gene of P. falciparum in 548 samples from children aged 2.5 or 24 months. Parasite prevalence in matched samples was compared using Mcnemar’s test and agreement of positive results quantified as Kappa scores. Parasite prevalences between different age groups were compared by Fisher’s test. Results from analyses by thick film microscopy were also available from children at 24 months and their correlation to each qPCR method examined by the Spearman’s test. Finally the association of P. falciparum infection with the incidence of multiple malaria episodes from contact to 24 months of age was evaluated using negative binomial regression.ResultsThese analyses showed that qPCR from whole blood detected approximately 3-fold more cases of infection than plasma qPCR. Both qPCR methods agreed well with each other although qPCR from plasma had a greater agreement with microscopy (96.85%) than did qPCR from blood (69.7%). At 24 months the prevalence of infection detected by all methods was associated with anaemia (p < 0.05).ConclusionsThe data presented here demonstrates that low levels of parasitaemia are better detected by qPCR using parasite DNA from whole blood than from plasma. However plasma samples provide a viable substitute when parasite smears are unavailable.

Highlights

Estimation of Plasmodium falciparum parasitaemia can vary with the method used and time of sampling
More recently other reports have demonstrated that quantitative PCR on DNA extracted from blood can be used to differentiate between species of Plasmodium [4] and can be a more sensitive method to quantify parasitaemia than microscopy of thick smears [5,6]
A total of 548 samples were analysed by Quantitative real time PCR (qPCR) on DNA extracted from whole blood on filter paper and plasma

Summary

Introduction

Estimation of Plasmodium falciparum parasitaemia can vary with the method used and time of sampling. More recently other reports have demonstrated that quantitative PCR (qPCR) on DNA extracted from blood can be used to differentiate between species of Plasmodium [4] and can be a more sensitive method to quantify parasitaemia than microscopy of thick smears [5,6]. The performance of qPCR on frozen plasma samples was compared with qPCR on whole blood collected onto filter paper to detect and quantify relative parasite levels in young children from Mozambique. This was with the aim of providing information on the relative utility of plasma samples where whole blood or blood smears are unavailable. Blood smears at 24 months were available for evaluation of parasitaemia by microscopy

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Discussion

Conclusion