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

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.

Malaria Diagnostics in Clinical Trials

“…However, for studies using archival material or in which whole blood or DBS are unavailable, testing of serum or plasma may provide useful data, albeit with less sensitive detection, than for whole blood. 106 Urine and saliva from malaria-infected persons contain detectable amounts of plasmodial DNA. Thus, this approach could be used as an alternative to microscopy or RDTs at sites that have established PCR capacity.…”

“…At the lowest parasitemias, sampling error will be a common cause of a false-negative result, and the prevalence of infection at sub-molecular parasitemias cannot be estimated. Finally, although there are reports of molecular diagnosis of malaria infection from urine and saliva, [103][104][105] plasma, 106 and serum 107 (as described later in this report), whole blood remains the most common sample type used for definitive diagnosis because the parasites reside in erythrocytes.…”

“…The literature contains several reports of PCR performed on serum or plasma samples. 106,107 Although parasite DNA can be detected in these sample types, the extra-hepatic parasite lives in erythrocytes and prospective malaria clinical trials are advised to design trials to use whole blood if available. However, for studies using archival material or in which whole blood or DBS are unavailable, testing of serum or plasma may provide useful data, albeit with less sensitive detection, than for whole blood.…”

Molecular Testing for Plasmodium falciparum by Use of Serum or Plasma and Comparison with Microscopy and Rapid Diagnostic Testing in Febrile Nigerian Patients

“…While traditional microscopy remains the primary malaria diagnostic method worldwide, malaria rapid diagnostic tests (mRDTs) are also being deployed for confirmation of suspected malaria cases. However, nucleic acid amplification tests (NAATs) for malaria, which have proven more sensitive than microscopy or mRDTs, are not routinely performed (3,4). Therefore, it is possible that estimates of overtreatment may be inflated due to the relatively poor sensitivity of microscopy for malaria diagnosis.…”

“…Therefore, it is possible that estimates of overtreatment may be inflated due to the relatively poor sensitivity of microscopy for malaria diagnosis. Although Plasmodium nucleic acids have been detected in serum and plasma, there is little published data describing the performance of malaria NAATs using these specimen types (4)(5)(6)(7). Rather, most studies have used whole blood or dried blood spots (DBS) (8)(9)(10)(11)(12).…”