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


METHODS
A 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.


RESULTS
The 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.


CONCLUSIONS
Large 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.
Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine (LSHTM), Keppel St, London, WC1E 7HT, UK.
Khalid B
KB
Beshir
Rachel L
RL
Hallett
Alice C
AC
Eziefula
Robin
R
Bailey
Julie
J
Watson
Stephen G
SG
Wright
Peter L
PL
Chiodini
Spencer D
SD
Polley
Colin J
CJ
Sutherland
D000962:Antimalarials / Q000494:pharmacology*; D016054:DNA, Protozoan / Q000032:analysis* / Q000235:genetics; D017125:Genes, Protozoan; D006801:Humans; D021261:Parasitic Sensitivity Tests / Q000379:methods; D010961:Plasmodium / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012358:RNA, Transfer, Met / Q000235:genetics
D000962:Antimalarials; D016054:DNA, Protozoan; D012358:RNA, Transfer, Met