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

Beshir KB, Hallett RL, Eziefula AC, Bailey R, Watson J, Wright SG, Chiodini PL, Polley SD, Sutherland CJ - Malar. J. (2010)

Bottom Line: Studies in Southeast Asia suggest some patients exhibit an extended parasite clearance time in the three days immediately following treatment with artesunate monotherapy.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.The duplex qPCR method tested may fulfil these criteria, and should now be evaluated in such field studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine (LSHTM), Keppel St, London, WC1E 7HT, UK.

ABSTRACT

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.

Show MeSH

Related in: MedlinePlus

Best fit log-linear parasite clearance curves for each patient. The graph shows the Parasite Reduction Ratio (PRR) for each anti-malarial treatment, estimated from qPCR data. PRR is the ratio in parasite density between admission and 48 hours post-treatment. R2 (coefficient of determination) reflects the goodness of fit of the log linear regression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2992070&req=5

Figure 3: Best fit log-linear parasite clearance curves for each patient. The graph shows the Parasite Reduction Ratio (PRR) for each anti-malarial treatment, estimated from qPCR data. PRR is the ratio in parasite density between admission and 48 hours post-treatment. R2 (coefficient of determination) reflects the goodness of fit of the log linear regression.

Mentions: The natural log of relative parasite abundance against time was plotted for each patient and fitted well with a simple linear model. Using this model of the parasite clearance dynamics in each patient, we again calculated PCT (with clearance defined as parasite density at 10-4% that of the starting sample, i.e. a reduction of 106-fold), PRR48 h and PCT95 parameters (Figure 3). These are compared to parameter values derived directly from microscopy and qPCR data in Table 3. For patients 1, 2 and 4 there is good agreement between the PRR48 h and PC95 parameter values estimated directly from microscopy or qPCR. Direct observation of PCT by qPCR was not possible for these patients, as all were parasite positive in this assay at the time of discharge, even though negative by microscopy. However, clearance time estimates derived from extrapolating the log-linear model to 10-4% of starting parasitaemia also agreed well with microscopic observations, and this was true whether microscopic or qPCR data were used to derive the line of best fit (Table 3). The one exception was patient 3, in that an observed clearance time by microscopy of 120 h in this individual, and a log-linear extrapolation from these data of 121 h, contrasts with a much longer clearance time of 183 h from qPCR data extrapolated to 10-4% of starting parasitaemia. This suggests that caution is needed in applying this methodology to estimate PCT, as opposed to reduction ratios, in patients with severe malaria presenting as hyperparasitaemia.


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

Beshir KB, Hallett RL, Eziefula AC, Bailey R, Watson J, Wright SG, Chiodini PL, Polley SD, Sutherland CJ - Malar. J. (2010)

Best fit log-linear parasite clearance curves for each patient. The graph shows the Parasite Reduction Ratio (PRR) for each anti-malarial treatment, estimated from qPCR data. PRR is the ratio in parasite density between admission and 48 hours post-treatment. R2 (coefficient of determination) reflects the goodness of fit of the log linear regression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992070&req=5

Figure 3: Best fit log-linear parasite clearance curves for each patient. The graph shows the Parasite Reduction Ratio (PRR) for each anti-malarial treatment, estimated from qPCR data. PRR is the ratio in parasite density between admission and 48 hours post-treatment. R2 (coefficient of determination) reflects the goodness of fit of the log linear regression.
Mentions: The natural log of relative parasite abundance against time was plotted for each patient and fitted well with a simple linear model. Using this model of the parasite clearance dynamics in each patient, we again calculated PCT (with clearance defined as parasite density at 10-4% that of the starting sample, i.e. a reduction of 106-fold), PRR48 h and PCT95 parameters (Figure 3). These are compared to parameter values derived directly from microscopy and qPCR data in Table 3. For patients 1, 2 and 4 there is good agreement between the PRR48 h and PC95 parameter values estimated directly from microscopy or qPCR. Direct observation of PCT by qPCR was not possible for these patients, as all were parasite positive in this assay at the time of discharge, even though negative by microscopy. However, clearance time estimates derived from extrapolating the log-linear model to 10-4% of starting parasitaemia also agreed well with microscopic observations, and this was true whether microscopic or qPCR data were used to derive the line of best fit (Table 3). The one exception was patient 3, in that an observed clearance time by microscopy of 120 h in this individual, and a log-linear extrapolation from these data of 121 h, contrasts with a much longer clearance time of 183 h from qPCR data extrapolated to 10-4% of starting parasitaemia. This suggests that caution is needed in applying this methodology to estimate PCT, as opposed to reduction ratios, in patients with severe malaria presenting as hyperparasitaemia.

Bottom Line: Studies in Southeast Asia suggest some patients exhibit an extended parasite clearance time in the three days immediately following treatment with artesunate monotherapy.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.The duplex qPCR method tested may fulfil these criteria, and should now be evaluated in such field studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine (LSHTM), Keppel St, London, WC1E 7HT, UK.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus