Measuring the efficacy of anti-malarial drugs in vivo: quantitative PCR measurement of parasite clearance

Khalid B Beshir,Spencer D Polley,Rachel L Hallett,Robin Bailey,Alice C Eziefula,Julie Watson,Stephen G Wright,Peter L Chiodini,Colin J Sutherland

doi:10.1186/1475-2875-9-312

Abstract

BackgroundArtemisinin-based combination therapy, currently considered the therapy of choice for uncomplicated Plasmodium falciparum malaria in endemic countries, may be under threat from newly emerging parasite resistance to the artemisinin family of drugs. Studies in Southeast Asia suggest some patients exhibit an extended parasite clearance time in the three days immediately following treatment with artesunate monotherapy. This phenotype is likely to become a more important trial endpoint in studies of anti-malarial drug efficacy, but currently requires frequent, closely spaced blood sampling in hospitalized study participants, followed by quantitation of parasite density by microscopy.MethodsA simple duplex quantitative PCR method was developed in which distinct fluorescent signals are generated from the human and parasite DNA components in each blood sample. The human amplification target in this assay is the β tubulin gene, and the parasite target is the unique methionine tRNA gene (pgmet), which exhibits perfect sequence identity in all six Plasmodium species that naturally infect humans. In a small series of malaria cases treated as hospital in-patients, the abundance of pgmet DNA was estimated relative to the human DNA target in daily peripheral blood samples, and parasite clearance times calculated.ResultsThe qPCR assay was reproducibly able to replicate parasite density estimates derived from microscopy, but provided additional data by quantification of parasite density 24 hours after the last positive blood film. Robust estimates of parasite clearance times were produced for a series of patients with clinical malaria.ConclusionsLarge studies, particularly in Africa where children represent a major proportion of treated cases, will require a simpler blood sample collection regime, and a method capable of high throughput. The duplex qPCR method tested may fulfil these criteria, and should now be evaluated in such field studies.

Highlights

Artemisinin-based combination therapy, currently considered the therapy of choice for uncomplicated Plasmodium falciparum malaria in endemic countries, may be under threat from newly emerging parasite resistance to the artemisinin family of drugs
As the majority of Artemisinin-based combination therapy (ACT)-treated patients are free of parasitaemia by microscopy within 48 hours of treatment, many closely-spaced venous blood samples in catheterized in-patients have been taken in previous studies to obtain a useful estimate of Parasite Clearance Time (PCT) by this method [5,6]
Written informed consent was given by patients 1, 2 and 3 for additional biological studies of malaria parasites isolated from their blood samples to be carried out, according to a protocol approved by the University College London Hospital (UCLH) Research Ethics Committee

Summary

Introduction

Artemisinin-based combination therapy, currently considered the therapy of choice for uncomplicated Plasmodium falciparum malaria in endemic countries, may be under threat from newly emerging parasite resistance to the artemisinin family of drugs. Studies in Southeast Asia suggest some patients exhibit an extended parasite clearance time in the three days immediately following treatment with artesunate monotherapy This phenotype is likely to become a more important trial endpoint in studies of anti-malarial drug efficacy, but currently requires frequent, closely spaced blood sampling in hospitalized study participants, followed by quantitation of parasite density by microscopy. As the majority of ACT-treated patients are free of parasitaemia by microscopy within 48 hours of treatment, many closely-spaced venous blood samples in catheterized in-patients have been taken in previous studies to obtain a useful estimate of PCT by this method [5,6] This approach is not suitable for large clinical trials, in African settings, where the majority of participants are likely to be outpatients under the age of 10 years; such excessive sampling would be considered unethical in most such studies. Absolute quantitation of parasite density from dried filter-paper blood spots is difficult, as the volume of blood in each spot is usually not known, and the efficiency of DNA extraction and target amplification differs between paper types and even among individual patients [9]

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Results

Discussion

Conclusion