Malaria parasite clearance

Malaria infection starts with the inoculation of a small number of sporozoites (median number estimated to be about 10) by a probing female anopheline mosquito. These motile parasites pass to the liver within an hour. Having invaded hepatocytes they then begin a period of rapid asexual multiplication [4, 5], dividing approximately every 8 h until each infected liver cell contains thousands of merozoites. Intrahepatic pre-erythrocytic development can be inhibited by some anti-malarials (antifols, 8-aminoquinolines, atovaquone, KAF 156, DMB 265) and some antibiotics (e.g. azithromycin, tetracyclines). In Plasmodium vivax infections and in both species of P. ovale malaria a sub-population of sporozoites form dormant liver stages called “hypnozoites” which awaken weeks or months later to cause relapses of malaria [4]. The hypnozoites can be killed only by 8-aminoquinolines of the currently available anti-malarial drugs.

At the completion of pre-erythrocytic development and following hepatic schizont rupture the newly liberated merozoites enter the blood stream and promptly invade erythrocytes. Then the growing intraerythrocytic malaria parasites begin to consume the red cell contents. The complete life cycle in the red blood cells approximates one day for P. knowlesi, two days for P. falciparum, P. vivax and Plasmodium ovale (two species) and three days for Plasmodium malariae [4]. A small sub-population of asexual parasites may stop growing and dividing for days or weeks (“dormancy”) [6]. Parasite multiplication rates in non-immune patients in this early stage of infection, before the symptoms of malaria have developed, range typically from 6 to tenfold per cycle (30–50% efficiency), but sometimes reach 20-fold [5, 7‐9]. Initial multiplication rates are similar for P. falciparum and P. vivax. As a result, total parasite numbers in the blood rise exponentially from 10⁴ to 10⁵ in the first asexual cycle to reach 10⁸ after 3–4 cycles (i.e. 6–8 days for P. falciparum and P. vivax) (Fig. 1). One hundred million parasites in the body of an adult human corresponds with a blood parasite density of about 50/µL [5, 7] and this density is usually associated with the onset of fever and illness in non-immune subjects (a “pyrogenic density”) [10, 11]. The addition of a pre-erythrocytic liver development of 5.5–7 days plus 6–8 days of blood stage multiplication results in the usual incubation period of 11–15 days in falciparum or vivax malaria [10, 11]. People who have had multiple previous malaria infections acquire an antitoxic immunity (“premunition”) which results in higher parasite densities being tolerated without symptoms, although densities over 10,000/µL are usually associated with illness even in areas of high malaria transmission [4, 10‐12]. Immunity slows parasite multiplication and accelerates parasite clearance. In most infections after logarithmic parasite multiplication there is an abrupt reduction in parasite multiplication at high densities. Severe malaria is a consequence of a failure of the infecting parasites to stop multiplying [13].

A sub-population of the blood stage parasites commit to sexual development forming male and female gametocytes. This reduces the parasite multiplication rate. Commitment (switching) to sexual development occurs immediately in vivax malaria (which becomes infectious to mosquitoes at, or even below pyrogenic densities) whereas gametocytogenesis is delayed in falciparum malaria (Fig. 1) [14]. Switching increases with duration of infection, anaemia and other stresses to the parasite population such as partially effective anti-malarial treatment. In P. falciparum infections, the developing sexual stages sequester for about 7–10 days in venules and capillaries and particularly in the bone marrow before reentering the circulation as immature stage 5 gametocytes [15]. As a result, peak P. falciparum sexual stage densities typically occur approximately 10 days after peak asexual densities [15]. Gametocytes are cleared relatively slowly from the blood so they accumulate with respect to asexual parasites and can predominate in chronic infections. The gametocytes of P. falciparum malaria are relatively insensitive to most anti-malarial drugs (with the notable exception of the 8-aminoquinolines) whereas the gametocytes of the other human malaria parasites are considered as drug sensitive as their asexual counterparts [14, 16].

Most natural malaria infections are relatively synchronous so the temporal pattern of parasite density rise in untreated malaria is generally log linear with superimposed oscillations resulting from synchronous schizogony [5, 17] (Fig. 1). The total parasite biomass is the product of the blood volume and the parasite count except in falciparum malaria where, because of sequestration, the peripheral parasite count variably underestimates the total parasite numbers. Sequestration describes the process whereby some 12–18 h after merozoite invasion P. falciparum parasitized erythrocytes adhere to vascular endothelium and disappear from the circulation [1]. Once adherent they do not detach until schizont rupture and so the parasites do not reappear in the circulation until the next asexual cycle [18, 19]. This results in a sinusoidal wave form pattern of parasitaemia with sharp rises and falls in parasite density corresponding with schizogony and sequestration, respectively [5] (Fig. 1). In falciparum malaria, large numbers of parasitized erythrocytes accumulate in the placenta and splenic pooling of parasitized erythrocytes may be significant in patients with splenomegaly [2, 20].

Three independent processes contribute to the clearance of malaria parasites from the peripheral blood circulation;

In symptomatic malaria, there is usually one dominant normally distributed population of parasite ages [17]. Sometimes “two brood” infections may be observed where two distinct age populations are evident [21]. In uncomplicated malaria, the age distribution of parasites at presentation to medical attention is not random. This is probably because previous cycle schizogony causes a pulse release of pro-inflammatory cytokines which provokes treatment-seeking [22]. Patients with uncomplicated malaria typically present to medical attention with a predominance of young ring stage parasites in the peripheral blood smear indicative of recent schizont rupture [4]. In contrast among patients with severe falciparum malaria the predominant parasite stages in peripheral blood smears appear randomly distributed. Marked fluctuations in parasite density shortly after starting treatment may therefore occur as a natural consequence of the infection itself (Fig. 1). If the majority of parasites in the body are mature schizonts that have not yet ruptured, a sharp rise in parasite count may occur immediately after admission to hospital (these sudden parasitaemia rises also occur in uncomplicated malaria but go unnoticed because frequent parasite counts are seldom made in outpatients) [23‐25]. Sudden alarming rises in parasite density were more common following the start of quinine than are now seen after artesunate treatment of severe falciparum malaria. Conversely in a synchronous infection, in which large P. falciparum ring stage parasites predominate in the blood smear, there may be a sudden decline in parasite density as these parasites sequester, giving the false impression of an excellent response to the anti-malarial treatment [7].

In all forms of malaria, parasitized erythrocytes can adhere to other erythrocytes (rosetting). Plasmodium falciparum and P. knowlesi-infected erythrocytes agglutinate with each other at high densities, and P. vivax infected erythrocytes can bind to chondroitin sulphate A (a cytoadherence receptor in the placenta), but there is little sequestration in these infections [1]. Except in falciparum malaria all parasite stages of development are seen in peripheral blood smears.

Parasite clearance from the blood reflects the stage-specificity and intrinsic potency of the anti-malarial drugs used. The slowest parasite clearance rates are seen following treatment with antibiotics, (e.g. tetracyclines) [26, 27], where the predominant effect is seen the second and subsequent drug-exposed cycles. The most rapid rates are seen following the start of treatment with the artemisinin derivatives and the spiroindolones [23, 28, 29]. The “batting order” of anti-malarial activities (measured in terms of parasite clearance times) in susceptible vivax malaria is similar to that in susceptible falciparum malaria (Fig. 2) with two exceptions; sulfonamides are relatively ineffective in P. vivax, and primaquine has only very weak blood stage activity against P. falciparum [29, 30].

Although the parasite density may rise or fall suddenly after starting anti-malarial treatment, in most cases there is a lag phase before parasitaemia falls. Thereafter the decline is log-linear (i.e. clearance is a first order process) [31, 32]. Most anti-malarial drugs have relatively little effect on circulating malaria parasites and so the initial decline in parasite density results both from parasitised red cell sequestration and any ring stage parasite killing and removal [7]. The faster parasite clearance following chloroquine compared with quinine treatment of severe malaria [33] (before chloroquine resistance had emerged) was attributed to a greater effect on ring stage parasites. Parasite clearance is even faster with artemisinin derivatives and the initial lag phase is less evident (Fig. 3) [1, 32]. Rapid clearance results from drug damage to the circulating ring-stage parasites and their subsequent removal predominantly by the spleen [34]. This prevents cytoadherence [35] and the pathological consequences of sequestration, and it largely explains why artesunate reduces mortality substantially in severe falciparum malaria compared with quinine [36]. Artemisinin resistance manifests as loss of ring stage susceptibility and thus slower parasite clearance [37‐39]. The slope of the log-linear phase of parasitaemia reduction (or the derived half-life) is particularly useful for assessing resistance to the artemisinins in vivo [31, 32, 37, 38] (Fig. 4), and is the metric which correlates best with heritability (i.e. has the strongest genetic association) [40]. Artemisinin resistance is associated with mutations in the propeller region of the kelch protein [41]. Different mutations confer different levels of resistance (i.e. different mean parasite clearance half-lives: PC_1/2) [38].

A parasite clearance curve can be constructed from a series of frequent sequential parasite counts, comprising thin film counts at higher densities and thick film counts at lower densities (>50/µL) [31, 32, 38, 42]. Highly sensitive uPCR methods can now quantitate a parasitaemia accurately down to densities of approximately 20/mL. RNA measurement is even more sensitive but as there are changing numbers of transcripts per parasite genome during the asexual life cycle, accurate quantitation of parasitaemia from mRNA measurement is more challenging. As uPCR DNA quantitation is possible at parasitaemias well below the pyrogenic density it is now possible to assess therapeutic responses to anti-malarial drugs in challenge studies without the volunteers becoming ill, and also to follow treated symptomatic infections which later recrudesce and to treat them again before symptoms develop [43‐45].

In general, anti-malarial drugs have their greatest activity against mature trophozoites, the most metabolically active stage of asexual parasite development which precedes DNA replication [46, 47]. A possible exception is chloroquine against P. vivax [48]. Very young ring stages of P. falciparum appear disproportionately sensitive to artemisinins [49]. The damaged and dead parasites in circulating erythrocytes are cleared predominantly by the spleen, as part of its normative function in removing intraerythrocytic particulate matter, although the liver, bone marrow and other lymphoid tissue play an important secondary role in parasitized erythrocyte clearance [34, 50‐53]. In falciparum malaria, the sequestered mature trophozoites are killed in situ and then disintegrate slowly. They leave behind erythrocyte membranes adherent to the vascular endothelium, and sometimes trapped malaria pigment, in the once sequestered vessels which is observed in post-mortem brain smears and electron microscopy studies of patients who have died after days of anti-malarial treatment [1, 19, 54, 55]. Clearance of this material is performed by circulating phagocytes (monocytes and polymorphonuclear leukocytes) [56]. At high parasite densities intraleukocytic pigment is observed commonly in blood films, and in severe malaria increased numbers of pigment containing neutrophils (>5%) have prognostic significance [57].

The spleen plays a central role in the control and clearance of intraerythrocytic infections [50]. The spleen’s normal function is to remove senescent red cells and circulating foreign material such as bacteria or cellular debris (often termed “refuse collection” and “policing” activities, respectively) [52, 53]. The structure of the spleen is complex with two overlapping blood circulations—a rapid flow by-pass, called the fast closed circulation, which typically takes 90% of the splenic blood flow (100–300 mL/min in a healthy adult), and a slow-open circulation in which the blood is filtered through narrow inter-endothelial slits. This slow filtration allows the blood elements to be assessed for antibody coating and deformability. Abnormal cells which fail inspection and other particulate material are retained [52, 53, 58]. In malaria, the spleen enlarges rapidly, and is often palpable (i.e. ≥3 times enlarged), and clearance function increases [59‐66]. Pathology studies of fatal human malaria which have examined the spleen show marked accumulation of parasitized erythrocytes of all stages [1, 2, 20, 50, 67‐71]. Similar findings are reported in primate malaria [72]. Thus, the “activated” spleen retains parasitized red cells (including ring stage infected cells) and it removes parasites and parasitized cells. Splenectomy and splenic dysfunction increase the risk of severe malaria [50, 51, 71], and splenic hypofunction probably contributes to delayed parasite clearance in immunocompromized HIV infected patients receiving anti-malarial treatment [71, 73, 74]. In endemic areas splenomegaly in childhood is used a measure of malaria transmission intensity [4]. There are three processes whereby the spleen can remove malaria parasites.

Despite enormous research investment and effort immunity to malaria is still poorly understood. In general terms, the acute malaria infection is contained by non-specific host-defence mechanisms including splenic activation and fever (which inhibits schizogony). Later more specific humoral and cellular immunity control and finally eliminate the infection. After weeks of illness in untreated infections parasitaemia is eventually reduced to levels which are tolerated with few or no symptoms. Untreated malaria parasitaemia can persist at low densities for months or years [98]. In malaria-endemic areas, where people are infected frequently, most infections are controlled at densities causing little or no symptoms, so some infections persist for weeks or months and many self- cure [4]. Illness results from infections to which the individual has insufficient immunity to control parasite multiplication [99]. In areas of higher transmission, this is most likely in young children who have had few or no previous infections. In older children and adults rapid mobilization of both non-specific and specific host-defence mechanisms usually results in prompt resolution of the infection—even without anti-malarial treatment. As a result “immunity” complements the effects of anti-malarial drugs, accelerating parasite clearance and augmenting cure rates [100‐103]. Failing drugs (i.e. anti-malarials to which resistance has developed) always perform much better in semi-immune patients. Acquired immunity explains why cure rates are always higher in adults and older children in endemic areas and why anti-malarial treatment efficacy assessments in high transmission settings should always include young children [7]. The magnitude of the effect of immunity on parasite clearance can be assessed by comparing parasite clearance rates in drug sensitive infections with similar drug exposure between high transmission and low transmission areas [32, 100], by assessing the effect of age on parasite clearance within an area of moderate or high transmission [101], or directly by correlating parasite clearance rates with malaria antibody titres [104]. In a recent large study the relationship of parasite clearance to titres of antibodies specific to 12 P. falciparum sporozoite and blood-stage antigens was assessed. P. falciparum antibodies were associated with significantly faster PC_½ values but the effects were relatively small; maximum shortening <40 min [104]. Immunity also reduces parasite multiplication (e.g. merozoite agglutinating antibodies) but this contributes relatively little to measures of immediate drug effect such as parasite clearance half-lives (PC_1/2). In the largest assessment to date the effect of age on parasite clearance following treatment with artemisinin derivatives was estimated in a subset of 3208 patients from areas without artemisinin resistance. Young children cleared parasites more slowly than older patients: PC_1/2 was 11.3% (95% CI 2.6–20.8, p = 0.010) longer in infants aged <1 year and 9.4% (95% CI 3.5–15.7, p = 0.002) longer in children aged 1–4 years compared to older patients. Overall PC_1/2 values were about 12 min faster in Africa than in Asia, where transmission is generally lower [101].

There is evidence from both clinical and laboratory studies that asexual blood stage parasites may become temporarily inert or dormant and so survive therapeutic concentrations of anti-malarial drugs. Dormancy is observed particularly following treatment with artemisinin derivatives [6, 105, 106] although it is unclear if the effect is a result of non-lethal cell damage or interference with cell cycling [107] (Fig. 7). It has been suggested that artemisinin resistance reflects an increased propensity for dormancy, although clinical and laboratory studies are more indicative of reduced ring stage artemisinin susceptibility [6, 37, 38, 107‐109]. The very high efficacy of ACT outside areas of artemisinin resistance suggests that dormant forms (or more likely these parasites when they wake) do not survive the residual concentrations of partner drugs. In general, dormant parasites are present at densities below the level of microscopy detection, although they may account for some of the “tail” in the parasite clearance curve observed particularly following the treatment of high parasitaemia infections, and they probably contribute significantly to persistent low density uPCR positivity (Fig. 7). The factors associated with dormancy, the metabolic state of the dormant parasites, and their natural history have yet to be characterized fully.

The sexual stages of P. falciparum are relatively insensitive to the anti-malarial drugs so they commonly persist after clearance of the asexual stages. Gametocyte densities in falciparum malaria reflect the balance between formation, release from sequestration, and clearance. Dormant forms which “awaken” can also presumably form gametocytes contributing to apparent slow gametocyte clearance. Gametocytes are readily distinguishable by microscopy, but as for dormant parasites, are indistinguishable from asexual parasites by quantitative PCR for DNA. Both therefore contribute to an apparent slow terminal elimination phase of parasite DNA in blood (Fig. 7). Gametocyte clearance also appears to be a first-order process although assessment of the rates of gametocyte clearance is often compromized by their low densities which results in greater errors in determining slopes (Fig. 7). Gametocyte clearance rates derived from microscopy over a few days following drug treatment may have underestimated the true clearance times of drug-unaffected gametocytes [110, 111]. The persistence of low density P. falciparum gametocytaemia for weeks after successful treatment of the asexual stage infection is not compatible with current estimates of either drug effects against early stage gametocytes or clearance times. Treatment with primaquine (or methylene blue) leads to rapid P. falciparum gametocyte clearance [112]. Gametocyte clearance underestimates drug effects in reducing infectivity. The majority of gametocytes in the circulation are female, yet the most anti-malarials have greater effects on male gametocytes [113]. Sterilization precedes gametocyte clearance. This temporal discrepancy is greatest with the 8-aminoquinolines which sterilize P. falciparum infections within hours but the gametocytes take days to clear [16]. Finally, it should be noted that drugs such as antifols and atovaquone may prevent zygote formation in the mosquito without affecting gametocyte clearance [114‐116].

Intra-host models of malaria infection have been developed to help characterize anti-malarial drug effects and hopefully guide treatment recommendations and deployment strategies. Anti-malarial drugs are usually modelled to kill parasites by a concentration-dependent process that is first order whilst anti-malarial drugs exceed minimum parasiticidal concentrations (MPC) [23, 89, 90, 108, 117‐119]. When anti-malarial drug concentrations fall below the MPC the effect is reduced and the decline in parasitaemia slows. The anti-malarial drug concentration when the parasite multiplication rate (PMR) is one can be termed a minimum inhibitory concentration (MIC) (Fig. 7) [23]. After anti-malarial blood concentrations fall below the MIC, the rise in parasite numbers is determined by the sub-MIC effects on multiplication and the effects of host immunity. Recrudescence occurs when parasitaemias reach densities detectable by microscopy (~50/µL). Stage specificity of anti-malarial drug action, second cycle effects, gametocyte switching, dormancy and increasing immunity can all be incorporated additionally in these PK-PD models.

As the anti-malarial drugs differ in their stage specificities of action [46, 47], the relationship between parasite stage distribution, pattern of drug exposure, parasite killing and clearance is complex. A weakness of many PK-PD models is that parasite killing by anti-malarial drugs is modelled as a single rate constant with a unit of time substantially less than the life span of the cell. A corollary is that for rapidly eliminated anti-malarials such as the artemisinins parasite damage is assumed to stop when drug concentrations decline. Thus, if a drug exceeded parasiticidal concentrations for 8 h (e.g. artemisinins) each day and this resulted in a 10,000-fold reduction in parasite density per asexual cycle, then it could be reasoned that ensuring the drug was present continuously at concentrations above the MPC would result in a 10¹² fold reduction per day (i.e. 10⁴ × 10⁴ × 10⁴). PK-PD modelling based on this assumption and saturated splenic clearance has concluded that giving artemisinin derivatives twice daily rather than once daily will “dramatically enhance and restore drug effectiveness” particularly in the management of artemisinin resistant falciparum malaria [89, 90, 108]. Clinical studies indicate that is untrue, presumably because single exposures provide near maximum effects, and the more mature sequestered stages remain susceptible in artemisinin resistance [102, 103, 120, 121]. Malaria parasites only need to be killed once in each generation. Current models of the time course of parasite killing may be oversimplifications. Another potential weakness is that parasite killing has been considered equivalent to or greater than parasite removal (mainly by the spleen), whereas it is likely that for drugs acting on ring stages (notably the PfATPase 4 inhibitors) drug affected viable parasites are removed, so splenic clearance rates may exceed the rates of killing of circulating parasites. For drugs which act on ring stage parasites the parasite clearance rate (or derived half-life) is currently the best in vivo measure of drug effect [31, 32, 37, 38, 40, 108, 122].

Current unresolved challenges in pharmacokinetic-pharmacodynamic modelling and anti-malarial dose optimization are how to structure models of parasite clearance, how to characterize the effects of host immunity and parasite “dormancy”, and our incomplete understanding of the behaviour of hypnozoites in P. vivax and P. ovale infections. Further improvements in parasite quantitation at low densities, particularly the quantitation of low density gametocytaemia, and development of methods which distinguish viable from dead or dying parasites will likely improve model fits, and thus their utility in predicting therapeutic responses. Two experimental approaches provide good characterization of initial anti-malarial responses; the human challenge model (in which “immunity” plays little or no role) [43, 44] and the laboratory model of immunodeficient mice transfused with human blood and infected with P. falciparum [123]. Identifying the anti-malarial MIC in an infection requires detailed individual prospective study of pharmacokinetics and sequential quantitation of parasitaemia using both microscopy and uPCR [117, 124]. The MIC is a critical PK-PD variable guiding dose optimization. It provides a method of calibrating in vitro susceptibility data from cultured parasites, and therefore marrying population pharmacokinetic data from different patient groups with susceptibility data from parasites all over the world to inform optimal dosing.