The parasite clearance curve
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Parasite clearance rates are important measures of anti-malarial drug efficacy. They are particularly important in the assessment of artemisinin resistance. The slope of the log-linear segment in the middle of the parasite clearance curve has the least inter-individual variance and is the focus of therapeutic assessment. The factors affecting parasite clearance are reviewed. Methods of presentation and the approaches to analysis are discussed.
KeywordsMalaria Falciparum Malaria Severe Malaria Parasite Density Artesunate
Factors affecting the parasite clearance curve
Frequency of sampling
In most therapeutic assessments parasite counts are taken once daily initially, or only on days 2 and 3. This is insufficient for definition of individual parasite clearance profiles, although it is enough for therapeutic comparisons, particularly if sample sizes are large enough. To characterize parasite clearance profiles adequately at least four data points are required (i.e. counts at least twice daily), and to define lag phases adequately counts at ≤ 6 hour intervals are required. Counts are made until negative (usually either 200 or 500 white cells are counted on the thick film). Many investigators check a further slide 12 to 24 hours later to "make sure".
Patients ill with acute falciparum malaria present with a range of parasitaemias. These initial parasite counts are approximately log-normally distributed. Counts vary over four orders of magnitude from approximately 100 to 1,000,000 parasitized erythrocytes/uL blood. In falciparum malaria, parasitized red cells circulate freely for only one third of the 48-hour asexual cycle. For the remainder they are sequestered in the venules and capillaries . The peripheral blood parasite count is therefore a variable underestimate of the total parasite burden [2, 3, 4]. Parasitaemias in infections with Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale are also approximately log-normally distributed, but very seldom exceed 100,000/uL. The zoonosis Plasmodium knowlesi, which has a quotidian cycle, may reach high parasite densities in humans and can be lethal. It is also not thought to sequester significantly. For these infections the peripheral blood parasite counts are an accurate reflection of the total burden. As parasite clearance is, for the most part, a first order process  then the higher the initial parasite density the longer counts will take to become undetectable (the parasite clearance time) (Figure 1).
Stage of development; schizogony
The parasite population varies in age of development over the 48-hour cycle (24 hours for P. knowlesi, 72 hours for P. malariae). The asexual cycle begins at merozoite entry to the erythrocyte and ends at schizont rupture. Based on microscopy staging most symptomatic infections are unimodal in age distribution, but some may be bimodal or multimodal [4, 19]. Developmental staging by microscopy is imprecise, providing age estimates within an approximate 4-6 hour time window. Whereas patients with severe malaria present with parasites at any mean age of development, in patients who present with uncomplicated falciparum malaria, young ring stages predominate in peripheral blood more frequently than would be expected by chance, i.e. random sampling of parasite age distributions. This well known observation, noted frequently in textbooks of tropical medicine, has been explained by synchronous schizogony resulting in a pulse release of pro-inflammatory cytokines, which provokes health-seeking behaviour . If schizogony leading to schizont rupture is occurring at the time of presentation and start of treatment then new parasites will be entering the circulation rapidly as treatment is given and, if the rate and the numbers of new parasites entering exceeds the clearance rate, the parasite density will rise. If ingress to the circulation balances egress then counts remain unchanged. None of the available drugs prevent schizogony if mature schizonts are present. Thus abrupt rises in parasitaemia may occur just after starting treatment of synchronous infections [21, 22, 23]. Infections may comprise more than one parasite genotype, although one usually dominates, and usually derive from more than one sporozoite and thus hepatic schizont. Maturation and hepatic schizont rupture may not be synchronous. The clearance curve therefore represents a weighted average of the individual intra- host parasite populations' stages of development and clearances.
Stage of development; sequestration
In Plasmodium falciparum infections mature parasites (pigment containing trophozoites and schizonts) are seldom seen in the peripheral blood because they cytoadhere to vascular endothelium and so are sequestered . This process begins at the large ring stage and may be accelerated by fever . As a consequence parasitaemia can fall abruptly in a synchronous infection simply as a result of cytoadherence consequent upon extensive sequestration . In such cases the predominant stages of parasite development in peripheral blood smears are large rings (i.e. the parasites which will soon sequester but have not yet done so) . Equally, as parasites are released from the rupture of sequestered schizonts in falciparum malaria, parasitaemia may rise considerably, and to a much greater extent than seen in the other malarias which do not sequester, and are not therefore hidden from the microscope. The predominant stages of parasite development in peripheral blood smears are therefore tiny rings. Reduction in fever may result in an apparent slowing of initial parasite clearance by delaying the onset of sequestration.
Most anti-malarial drugs affect predominantly the more mature trophozoite stages of parasite development [25, 26]. This results in clearance of the circulating parasitized erythrocytes in the so-called benign malarias, or in the case of falciparum malaria, death of the sequestered parasites . As the sequestered parasites are not seen in the peripheral blood film the consequences of their killing on peripheral blood parasite counts are delayed. Furthermore unless the anti-malarial achieves therapeutic concentrations immediately (which occurs only with parenteral artesunate) there is also a variable delay related to drug absorption (which particularly slow for intramuscular artemether and artemotil) or slow intravenous infusion (required for quinine). As a result the changes in parasitaemia that occur immediately (i.e in the hours) after starting anti-malarial treatment are largely those that would have happened anyway . Rises in parasitaemia result from schizogony and falls from sequestration . In uncomplicated malaria parasite counts are seldom taken more frequently than once daily and these early fluctuations in parasitaemia may go unnoticed. In severe malaria following treatment with quinine, which does not affect greatly the circulating ring stages , the net result in an individual patient is that parasitaemia may, rise, fall or plateau until the parasites begin to sequester (an interval of no more than 12 hours) depending on the mean age and distribution of ages of the infecting parasites at presentation [28, 29, 30, 31]. As a consequence the average profile in a patient series of parasite clearance times with quinine treatment is a brief down-sloping plateau followed by a log-linear fall. The sustained fall in parasitaemia results from some ring stage killing, but predominantly from the killing of sequestered parasites which cannot therefore mature and multiply to feed new young parasites into the circulation. For fully sensitive parasites chloroquine results in faster parasite clearance, which presumably reflects a broader stage-specificity of action compared with quinine .
Haemoglobinopathies and glucose - 6-phosphate dehydrogenase deficiency may augment to host defences against malaria but the most important contributors are the non-specific and the specific immune responses to malaria. Therapeutic responses in malaria are enhanced by immunity . Drugs which are clearly ineffective in non-immunes (usually children) may appear to be very effective in immune adults. Self-cure is usual in endemic areas even without anti-malarials. Antibody alone can be used to treat malaria [40, 41]. Even in low transmission areas, with little background immunity, parasite clearance rates vary substantially between individuals infected with genetically identical parasites who receive artemisinin combination treatment . This presumably reflects inter-individual differences in anti-malarial pharmacokinetics, splenic function and other non-specific host defences, and any acquired immunity. This remains difficult to characterize in malaria as there are still no good ex-vivo correlates of protective immunity . Nevertheless some generalizations are possible. As immunity increases parasite counts are lower, severe malaria is less common, and parasite clearance is accelerated so the slopes of parasite clearance curves become steeper. In endemic areas this is reflected in the differing clinical presentations and therapeutic responses with increasing age. Conversely as immunity declines, for example if transmission is reduced, then parasite clearance rates fall.
Presentation of data
Analysis of data
Parasite clearance is usually analysed to assess the suitability of new treatments or to investigate changes in anti-malarial drug susceptibility. It is the drug effect (pharmacodynamics; PD) that is of primary interest, so the analysis attempts to account for the other confounders, which are drug-independent. Drug exposure (pharmacokinetics; PK) should also be assessed to determine how much of the observed variance in parasite clearance can be accounted for by inadequate drug exposure. PK-PD relationships for anti-malarial drugs are still poorly characterized, and most reported anti-malarial drug trials do not have a PK component, often leaving their interpretation uncertain. Parasite clearance has been expressed in a number of ways. The parasite clearance time (PCT) is most widely used, but this is a function of the pre-treatment parasite count (Figure 1), and it is critically dependent on the accuracy of microscopy and the frequency with which blood slides are taken. Any stage specificity of drug action is not reflected in this variable as PCTs are typically between 1 and 3 days. This may not matter in uncomplicated malaria but in infections with high parasitaemia and in severe malaria the speed of initial response is the critical determinant of outcome. The times taken for parasitaemia to fall by 50% (PC50) and 90% (PC90) have been used as alternatives, which are less affected by these covariates, but still require extrapolation from clearance curves . The slope of the linear segment of the log parasitaemia versus time curve is a relatively robust variable as it is determined from a best fit to several data points well within the countable range, although it does not reflect fully stage specificity of drug action. The slope can be determined visually, using least squares regression, or by modelling. It remains to be decided what should be the criteria for defining the top of the slope (i.e. maximum value, or intersection of linear fit to the initial plateau, or modelled fit), and for the lowest densities that should be included at the tail of the curve .
The parasite clearance time is a useful measure of anti-malarial drug effect but it is imprecise and depends upon the initial parasitaemia. More detailed assessment of the parasite clearance curve is needed to assess in-vivo responses to the artemisinin derivatives. Research is in progress to define the optimum methods and threshold values, which identify artemisinin resistance. Standardization of methodologies for the assessment of anti-malarial drug resistance is a primary objective of the WorldWide Antimalarial Resistance Network (WWARN) and a WWARN "parasite clearance estimator" providing a standard method of analysing parasite clearance data is now available as a freely accessible on-line tool . This should facilitate comparison of in-vivo studies and help map the emergence and spread of resistance.
I am very grateful for advice from my colleagues in the Mahidol Oxford Research Unit. I am a Wellcome Trust Principal Fellow.
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