Assessment of therapeutic responses to gametocytocidal drugs in Plasmodium falciparum malaria
- Nicholas J White1, 2Email author,
- Elizabeth A Ashley1,
- Judith Recht1,
- Michael J Delves3,
- Andrea Ruecker3,
- Frank M Smithuis2, 4,
- Alice C Eziefula5,
- Teun Bousema5,
- Chris Drakeley5,
- Kesinee Chotivanich1, 7,
- Mallika Imwong1, 6,
- Sasithon Pukrittayakamee1, 7,
- Jetsumon Prachumsri8,
- Cindy Chu1, 9,
- Chiara Andolina1, 9,
- Germana Bancone1, 9,
- Tran T Hien10,
- Mayfong Mayxay11,
- Walter RJ Taylor1,
- Lorenz von Seidlein1,
- Ric N Price1, 12,
- Karen I Barnes13,
- Abdoulaye Djimdé14,
- Feiko ter Kuile15,
- Roly Gosling16,
- Ingrid Chen16,
- Mehul J Dhorda2, 17,
- Kasia Stepniewska17,
- Philippe Guérin17,
- Charles J Woodrow1, 2,
- Arjen M Dondorp1, 2,
- Nicholas PJ Day1, 2 and
- Francois H Nosten1, 2, 9
© White et al.; licensee BioMed Central. 2014
Received: 8 November 2014
Accepted: 29 November 2014
Published: 9 December 2014
Indirect clinical measures assessing anti-malarial drug transmission-blocking activity in falciparum malaria include measurement of the duration of gametocytaemia, the rate of gametocyte clearance or the area under the gametocytaemia-time curve (AUC). These may provide useful comparative information, but they underestimate dose-response relationships for transmission-blocking activity. Following 8-aminoquinoline administration P. falciparum gametocytes are sterilized within hours, whereas clearance from blood takes days. Gametocytaemia AUC and clearance times are determined predominantly by the more numerous female gametocytes, which are generally less drug sensitive than the minority male gametocytes, whereas transmission-blocking activity and thus infectivity is determined by the more sensitive male forms. In choosing doses of transmission-blocking drugs there is no substitute yet for mosquito-feeding studies.
The objective of anti-malarial drug treatment is to cure the malaria infection as rapidly, reliably and safely as possible . In endemic areas there is another important public health consideration and that is to stop the treated infection being transmitted to anopheline mosquitoes, and thereby to other people. Treating the asexual stage infection effectively prevents generation of young gametocytes and it also prevents recrudescences (which are associated with increased gametocyte carriage rates). This reduces the transmission potential of the treated infection . Plasmodium falciparum differs from the other malaria parasites of humans in that the emergence of gametocytaemia is delayed with respect to asexual parasitaemia. The anti-malarial drugs which are used to treat falciparum malaria also kill young developing sexual stage parasites but they have little or no activity against the mature transmissible gametocytes. Treated infections are an important source of transmission especially in areas of low, unstable malaria transmission. Artemisinin combination treatment (ACT), the first-line treatment for falciparum malaria, reduces gametocyte carriage more than do other anti-malarial treatments [3–6], but even ACTs do not eliminate mature transmissible P. falciparum gametocytes [3–9]. Preventing these patients’ infections from transmitting falciparum malaria requires treatment with a specific gametocytocide, and the only generally available drug is the 8-aminoquinoline, primaquine [6–8, 10]. Primaquine rapidly sterilizes the infection, thereby reducing its transmissibility . Given together with an effective treatment for the asexual stage parasites, only one dose is required. Historically this dose has been 0.5-0.75 mg base/kg . Primaquine taken with food is generally well tolerated. The main adverse effect of the 8-aminoquinolines is dose-dependent oxidative haemolysis in people with glucose-6-phosphate dehydrogenase (G6PD) deficiency [10, 11]. G6PD deficiency is very common in many malaria-endemic regions. In some areas concerns over safety in patients with G6PD deficiency have limited deployment of primaquine as a gametocytocide .
Dosing recommendations based on gametocytaemia; the Cochrane Review
In contrast, the Cochrane analysis of the published study concluded that addition of primaquine 0.25 mg/kg to quinine “did not demonstrate an effect on gametocytaemia” . This appears to be a mistake derived both from a mix-up between the reported effects of the lower and higher primaquine doses (median gametocyte clearance times were shorter, not longer, with the 0.25 mg/kg dose compared to the 0.5 mg/kg dose as shown in Figure 2), and also misreading or misinterpretation of a table describing proportions of patients who were gametocytaemic. Although the relevance of a seven-day primaquine regimen to single-dose gametocytocidal use is uncertain, the Cochrane analysis used these data to argue that a primaquine dose of 0.25 mg/kg had no effect on gametocyte clearance – which is incorrect.
Transmission of human malaria requires that the gametocytes are infectious to feeding anopheline mosquito vectors. Mosquito-feeding studies assess transmission-blocking effects directly and are therefore the most appropriate way to measure the pharmacodynamic effect for which the drug is prescribed. However, they are notoriously difficult, and as a consequence only few have been conducted . It is much easier to measure gametocyte densities over time in blood samples either by microscopy or by molecular methods, and so there are many more data on these indirect assessments [2–5, 7–9, 16, 17]. These indirect measures based on gametocyte densities in blood have been used in drug evaluations based on the assumption that their results agree closely with transmission-blocking activity and can therefore be substituted for direct assessments. But it is well known that gametocytaemia does not equate with infectivity [10, 18]. The generally sigmoid relationship between gametocyte density and mosquito infectivity is very variable with some patients infecting mosquitoes without detectable gametocytaemia and others with high densities not being infectious.
The ratio of female to male gametocytes is unequal in falciparum malaria with a strong female bias in most studies [20, 23, 24]. This is presumably because each male gametocyte can generate eight microgametes and can therefore potentially fertilize eight females. There is variation in reported female to male ratios in acute malaria with values of 3-5 to 1 being observed mainly [22, 24–30] (Figure 5). At lower densities in anaemic children with chronic infections, the female to male gametocyte ratios are reportedly lower , but at the higher densities in acute infections in which drug assessments are performed females consistently comprise >70% of the P. falciparum gametocytes. The biology of male and female gametocytes is very different. Since the early days of malariatherapy it was recognized that successful transmission of malaria to mosquitoes required fully mature gametocytes, and microgametocytes which readily exflagellated. Crucially it is these minority male (micro)gametocytes which are usually more drug sensitive . In 1928 Mollow described greater sensitivity of male gametocytes to the 8-aminoquinoline plasmochin (plasmoquine, pamaquine) the predecessor of primaquine . Shute and Maryon in their extensive experience of artificial infection (malariatherapy) studies noted the following for P. falciparum: “in our cases female gametocytes almost invariably make their appearance several days before males and throughout the period of their presence the females outnumber the males from between 5 and 20 to 1 or more, and then when the gametocytes begin to diminish it is the males which fall off in numbers, and the females appear to persist for a longer period in the peripheral blood” . Thus if a drug killed all the male gametocytes but none of the females, it would sterilize the infection but gametocyte density would fall by less than 30%.
Counting gametocyte densities
Serial asexual parasite counts in blood are used to assess therapeutic responses to antimalarial drugs. Asexual parasite clearance times are dependent on several factors in addition to the drug treatment including initial parasite counts, proportion of P. falciparum asexual parasites which are sequestered, immunity, and the counting methods used . These factors also affect gametocyte clearance assessments. However gametocyte densities are orders of magnitude lower in acute malaria so counting errors are correspondingly greater. Gametocyte densities are usually quantitated on thick blood films with males and females counted together. There is input to the circulation from sequestered immature gametocytes while dead gametocytes are cleared predominantly by the spleen, so in drug assessments gametocyte input needs to be deconvoluted from clearance. Artemisinins, and to a lesser extent other anti-malarials which act on blood stage parasites, mainly reduce the input into the circulation whereas primaquine (and other 8-aminoquinolines) accelerate clearance . Live gametocytes may circulate for many days (mean estimates 4.5 to 6.5 days) . Microscopy cannot distinguish reliably between younger Stage V gametocytes which have just entered the circulation and are not yet infectious and older Stage V gametocytes which are infectious. This explains why gametocytaemic patients with acute malaria may not infect mosquitoes when first assessed. Microscopy also cannot distinguish between live and dead gametocytes. Assessment of the sex ratio by microscopy requires experience, and is more difficult on thick films and after treatment with gametocytocidal drugs. Measurement of mRNA transcripts by qPCR methods is more sensitive than microscopy. The most widely used is the QT-NASBA (quantitative nucleic acid sequence-based amplification) based on detection of Pfs25 transcripts [8, 16, 36–38]. Importantly Pfs25, and its orthologues in rodent malarias, are expressed much more abundantly in female gametocytes. Indeed they appear to be female specific (although whether Pfs 25 is transcribed but not expressed in male gametocytes still needs to be determined) [32, 38, 39].
Gametocyte clearance assessment
Gametocyte clearance time estimates depend on the detection methods used. Times derived from the more sensitive QT-NASBA are obviously longer than those based on microscopy. Times derived from thick film counts per 1000 white cells will be longer than those per 200 white cells. Fitting a mathematical model of gametocyte dynamics to measurements of sub-microscopic gametocyte prevalence and density  to estimate of time to gametocyte clearance has also been used as a measure of drug effect . As for asexual parasite clearance estimates, the times to clearance of gametocytaemia are proportional to initial gametocyte densities and the quality and sensitivity of microscopy or the assay used. Whatever method is used the higher the initial density the longer it takes before densities fall below the level of detection  (Figure 6).
Male gametocytes are usually more drug sensitive than females , and it is very likely therefore that they are cleared more rapidly than female gametocytes [33, 34] (Figure 6). Total gametocyte counts by microscopy reflect the predominant female gametocyte population (~70-90% of the total) – and Pfs25 QT-NASBA provides estimates predominantly or only for female gametocytes. If the male gametocytes are more drug sensitive than the females, and they are cleared more rapidly, then the gametocytaemia AUC is determined largely, and the proportions of patients with gametocytaemia on day 8 and the gametocyte clearance times are determined solely by the less drug-sensitive females (see Figure 6).
It is not known how rapidly dead gametocytes are cleared from the blood.
The area under the gametocytaemia time curve (AUC), whilst providing a model independent measure, assumes that density and time are equally important in terms of infectivity to mosquitoes, which clearly they are not. Without logarithmic transformation a gametocyte density of 1,000/uL for three days provides the same AUC as a density of 100/uL for 30 days. With logarithmic transformation this becomes 4.5 days. High gametocyte densities in patients presenting with acute symptomatic falciparum malaria are often not infectious at all, comprising the first wave of young Stage V gametocytes, whereas low densities later on are usually infectious [18, 34, 43]. In the example above gametocyte carriage at transmissible densities for 30 days provides ten times more opportunity for mosquitoes to bite than carriage for three days.
8-aminoquinolines sterilize malaria infections within hours, but gametocyte clearance does not start to increase for > one day  (Figure 7), yet the first day post-treatment may contribute a significant proportion of the total AUC.
Use of the AUC also assumes a linear relationship between gametocyte density and infectivity whereas in reality this relationship is sigmoid [10, 18, 43, 44]. Unusual patients with very high gametocyte densities contribute disproportionately to the AUC estimate, although their potential infectivity is saturated.
Cochrane review analysis of gametocytaemia in drug comparisons; the AUC
The Cochrane review  used the following formulas for the area under the gametocyte –time curve.
AUC (days 1 to 15) = ((8-1)*(G1 + G8)/2) + ((15-8)*(G15 + G8)/2)/14 for days 1 through 15.
AUC (days 1 to 29) = ((8-1)*(G1 + G8)/2) + ((15-8)*(G15 + G8)/2) + ((29-15)*(G29 + G15)/2)/28 for days 1 through 29.
AUC (days 1 to 43) = ((8-1)*(G1 + G8)/2) + ((15-8)*(G15 + G8)/2) + ((29-15)*(G29 + G15)/2) + ((43-29)*(G43 + G29)/2)/42 for days 1 through 43
where Gx = mean gametocyte density on day X (estimated using data from all participants still enrolled on day X). The log(10) AUC values were estimated using geometric mean gametocyte density.
Gametocyte sterilization is rapid but killing is slower.
Clearance of dead gametocytes from the blood is slow.
8-aminoquinolines affect predominantly the minority male gametocyte population .
Although sub-microscopic densities of gametocytes are an important source of malaria transmission [7, 44, 50], this is usually from asymptomatic individuals at quasi-steady state parasitaemias. In symptomatic patients it seems reasonable to predict that soon after gametocyte densities fall below the level of microscopy detection the treated patient becomes non-infectious – provided that the asexual infection is cured (i.e., that there is not continued low-level asexual stage multiplication generating new gametocytes), and there is no re-infection. If one drug clears gametocytaemia more rapidly than another, then it seems reasonable to conclude that it will also reduce transmissibility more. More information is needed on these points. However for the reasons given above determining the dose-response relationship for a drug (such as primaquine) based on indirect measures is likely to provide a significant underestimate of the relationship for transmission-blocking activity.
8-aminoquinoline effects on infectivity to mosquitoes
In 1927, shortly after its development, the 8-aminoquinoline plasmoquine (the predecessor of primaquine) was shown to clear gametocytaemia more rapidly than asexual stages in falciparum malaria . By 1929, studies conducted in Panama showed that plasmoquine rapidly reduced the infectivity of P. falciparum to anopheline mosquitoes [55, 56]. The transmission-blocking effect (assessed by reduction in mosquito oocyst counts) preceded the effects on gametocytaemia. In subsequent mosquito-feeding studies plasmoquine doses as low as 10 mg provided rapid and potent transmission-blocking activity [53, 57–60]. Primaquine replaced plasmoquine 65 years ago because it was safer and more potent; it was three times more active against pre-erythrocytic stages, four to six times better in radical curative activity against vivax malaria, and was half as toxic. The transmission-blocking activities of the two drugs were never compared but, given their structural and pharmacological similarity, it seems very likely that the pharmacodynamic effects would also be similar and very unlikely primaquine would be less active than plasmoquine. In 1959, Young reported the effects of an adult primaquine dose of 3 mg per day (0.05 mg/kg) in two malariatherapy patients. This very small dose abolished P. falciparum infectivity to mosquitoes by the third and fifth days of treatment, which was before any decline in gametocytaemia  (Figure 9). A recent review found data from 158 individual gametocytaemic subjects from different studies in which infectivity in mosquito-feeding studies was assessed both by oocyst and sporozoite production 24 and 48 hours after drug exposures . Of these, 31 subjects received plasmoquine (before 1950) and 127 received primaquine (69 together with an artemisinin derivative). These studies show clearly that both plasmoquine and primaquine rapidly and potently reduce the infectivity of P. falciparum within hours (Figure 1), whereas there is a lag phase >24 hours before gametocyte clearance starts (Figure 7) .
Assessment of anti-malarial drug transmission-blocking activity in vivo is problematic. Mosquito-feeding studies assess directly the effect for which the drug is prescribed (i.e., to block transmission of malaria) but they are difficult and laborious, and there are few data as a consequence. Fortunately there is renewed interest in these assessments and more information to guide evaluations of transmission-blocking effects drugs and vaccines will soon become available. Meanwhile indirect measures based on quantitation of gametocytaemia provide a guide to the comparative activity of different drugs or different drug dosages - but they make a number of assumptions. Critically current gametocyte clearance measures probably underestimate significantly the inhibitory effects of 8-aminoquinolines on infectivity. This compromises dose-response assessments both for the time of onset and the magnitude of transmission-blocking activity. The unique studies of Rieckmann et al. in which volunteers were infected with P. falciparum and managed to a steady state of parasitaemia highlights the discrepancies between total gametocytaemia, mosquito oocyst development, sporozoite formation, and the infectivity of these mosquitoes to naïve volunteers [52, 61] (Figure 8). Early observations on the greater sensitivity of male gametocytes to plasmoquine (pamaquine) , the more rapid clearance of male gametocytes , and the recent study showing large differences between the drug susceptibility of male and female gametocytes  provide further reason to be cautious in interpreting drug comparisons based on total gametocytaemia quantitated by microscopy or predominantly female gametocytaemia assessed by quantitation of Pfs25 transcripts. The degree of underestimation by indirect assessments is unclear as there have been few simultaneous comparisons. A comparison of different studies (albeit with different patient groups, designs, drug dosages, etc.) in P. falciparum infections (summarized in Figures 1 and 7) suggests a significant difference. If infectivity is determined by the susceptibility of male gametocytes, then this comparison would suggest that male gametocytes may be more than three times more susceptible to primaquine than females (Figures 6 and 10) but further studies are required to determine whether these predictions are correct. On the other hand, it seems very unlikely that these indirect assessments based on gametocyte clearance overestimate transmission-blocking activity so they do set an upper limit for a dose-response relationship which may be useful.
More direct assessments of drug effects on male and female gametocytes individually such as the P. falciparum dual gamete formation assay (Pf DGFA) are an advance [62, 63], and histochemical methods may distinguish live from dead gametocytes, but for the present for dose-response assessments there is no substitute for mosquito-feeding studies (although close attention must be given to the specific design of these assays to ensure that the outcomes can be interpreted and compared usefully) [45–49]. Primaquine is the only generally available drug, which rapidly and reliably reduces the transmissibility of P. falciparum malaria infections from patients presenting with patent gametocytaemia. Unfortunately the 8-aminoquinolines require hepatic bioactivation which compromises ex vivo assessments. Characterizing sex-specific markers and assessing the pharmacokinetic-pharmacodynamic relationship for primaquine’s gametocytocidal activity on male and female gametocytes in vivo is a research priority. Ideally dose-response assessments should be performed by comparing the infectiousness of gametocytaemic blood to anopheline mosquitoes before and after different doses, preferably with assessment of both oocyst and sporozoite formation, plasma primaquine concentration measurement (and CYP2D6 genotyping) . Given the pharmacokinetic properties and the documented rapidity of onset (particularly for inhibition of sporozoite formation) testing before and eight to 24 hours after dosing would reduce variability in the feeding mosquitoes.
Despite their difficulty, mosquito-feeding studies still remain the ‘gold standard’ for the assessment of transmission-blocking dose-response relationships. More information is needed on the relative susceptibility of male and female gametocytes and the kinetics of male and female gametocyte killing, together with direct comparisons with infectivity assessments, before indirect measures can be used confidently to make dosage recommendations.
In appreciation of Bob Sinden and his extensive contribution to our understanding of malaria transmission. NJW, EAA, JR, FMS, MI, KC, SP, CC, GB, CA, CJW, WRJT, LvS, AMD, NPJD, and FHN are supported by the Wellcome Trust (Programme Grant 089179) and were part of the Wellcome Trust Mahidol University Oxford Tropical Medicine Research Programme funded by the Wellcome Trust. MJD, CD, TB, RG and IC are supported by the Bill and Melinda Gates Foundation (MD: OPP1043501, CD: OPP1034789, TB: OPP1024438 and OPP1034789, RG and IC A115501); AR is supported by the Medicines for Malaria Venture (MMV 08/2800). The Wellcome Trust also supports ACE (090558), CD (091924) and RNP (091625). MJD, KS and PG are supported by the WorldWide Antimalarial Resistance Network.
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