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Determinants of relapse periodicity in

Plasmodium vivax malaria
White

White Malaria Journal 2011, 10:297
http://www.malariajournal.com/content/10/1/297 (11 October 2011)

White Malaria Journal 2011, 10:297
http://www.malariajournal.com/content/10/1/297

REVIEW Open Access

Determinants of relapse periodicity in
Plasmodium vivax malaria
Nicholas J White

Abstract
Plasmodium vivax is a major cause of febrile illness in endemic areas of Asia, Central and South America, and the
horn of Africa. Plasmodium vivax infections are characterized by relapses of malaria arising from persistent liver
stages of the parasite (hypnozoites) which can be prevented only by 8-aminoquinoline anti-malarials. Tropical P.
vivax relapses at three week intervals if rapidly eliminated anti-malarials are given for treatment, whereas in
temperate regions and parts of the sub-tropics P. vivax infections are characterized either by a long incubation or a
long-latency period between illness and relapse - in both cases approximating 8-10 months. The epidemiology of
the different relapse phenotypes has not been defined adequately despite obvious relevance to malaria control
and elimination. The number of sporozoites inoculated by the anopheline mosquito is an important determinant
of both the timing and the number of relapses. The intervals between relapses display a remarkable periodicity
which has not been explained. Evidence is presented that the proportion of patients who have successive relapses
is relatively constant and that the factor which activates hypnozoites and leads to regular interval relapse in vivax
malaria is the systemic febrile illness itself. It is proposed that in endemic areas a large proportion of the
population harbours latent hypnozoites which can be activated by a systemic illness such as vivax or falciparum
malaria. This explains the high rates of vivax following falciparum malaria, the high proportion of heterologous
genotypes in relapses, the higher rates of relapse in people living in endemic areas compared with artificial
infection studies, and, by facilitating recombination between different genotypes, contributes to P. vivax genetic
diversity particularly in low transmission settings. Long-latency P. vivax phenotypes may be more widespread and
more prevalent than currently thought. These observations have important implications for the assessment of
radical treatment efficacy and for malaria control and elimination.

Introduction after resolution of the primary infection. In endemic
In endemic areas of Asia, Oceania, Central and South areas relapse of vivax malaria is a major cause of malaria
America, and in the horn of Africa Plasmodium vivax in young children, and an important source of malaria
malaria is a major cause of morbidity. It is an important transmission. Relapse also occurs in Plasmodium ovale
contributor to early pregnancy loss and reduced birth infections and in several of the simian malarias, notably
weight which increases mortality in infancy [1,2]. Plas- Plasmodium cynomolgi, which has often been used as an
modium vivax is a sophisticated and resilient malaria animal model of vivax malaria. The factors which con-
parasite which was once prevalent over much of the trol relapse and determine their remarkable periodicity
inhabited world. It has receded from North America, are not known.
Europe and Russia, but in the tropics vivax malaria
remains a major cause of childhood illness. In most History
endemic areas, P. vivax cohabits with Plasmodium falci- The history of clinical research on vivax malaria con-
parum. Mixed infections with the two species are com- tains a wealth of valuable information that is not widely
mon. P. vivax is more difficult to control and eliminate known or recognised. Most studies were conducted over
than P. falciparum because of its tendency to relapse fifty years ago and are not readily accessible to the mod-
ern reader. Indeed more may have been forgotten than
Correspondence: nickw@tropmedres.ac has been learned about vivax malaria in recent years.
Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical
Medicine, Mahidol University, 420/6 Rajvithi Rd, Bangkok, 10400, Thailand The tendency of malaria infections to recur was well

© 2011 White; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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known since Roman times. From the 1630s onwards a observations describing latencies of some eight to nine
specific treatment (Cinchona bark) was available for months were complemented by Korteweg’s detailed and
agues (although for most of those infected it was unaf- painstaking prospective epidemiological observations
fordable). This treatment was often followed by frequent over more than two decades in the village of Wormev-
relapses of the periodic fever, and so opinions differed eer in The Netherlands. Celli had long surmised that
widely on its efficacy. Following Laveran’s discovery of the spring wave of benign tertian (P. vivax) malaria in
the blood parasite that caused malaria in 1880 [3], Northern Italy resulted from relapse [13,14] but it was
understanding of malaria epidemiology, pathology, and Korteweg who provided convincing evidence that the
treatment was placed on a rational basis. In 1885, Golgi vivax malaria which emerged in the early summer had
in Pavia distinguished the parasites responsible for ter- been acquired in the autumn of the previous year
tian and quartan fevers [4]. In 1890, P. vivax was identi- [15-19] (Figure 1). The considerable vivax malaria
fied as a separate species by Grassi and Feletti [5], experience of the First World War (in the Balkans,
although debate continued into the early 1920s as to Mesopotamia, and the Jordan valley), the secondary
whether there were indeed separate human malaria spe- cases in England which followed the year after the
cies, or just one single polymorphic species [6]. Studies return of infected soldiers, studies in American soldiers
of the time often tended to consider the responses of serving in Panama and the Philippines, studies in British
the benign (vivax) and malignant (falciparum) tertian soldiers serving in India, studies in Japanese soldiers
malarias together. The Dutch physician Pel is generally who had invaded China, and further epidemiological
credited with the first postulation of an exoerythrocytic observations and individual case reports in Europe and
stage of malaria in 1886 [7]. In 1897, the American phy- the United States all pointed to a disease which could
sician WS Thayer gave a very clear description of a relapse within two months of stopping quinine treat-
long-latency relapse of malaria 21 months after the ment, but also tended to relapse some eight to ten
initial attack in a physician who had probably not been months later (Figure 2). Until the 1920s it was common
re-exposed between the two events [8]. Thayer and to recommend an eight-week course of quinine treat-
Bignami (in Italy) [9] both surmised that relapses of ment for malaria [20]. Despite this protracted treatment
malaria resulted from a “spore” deposited in the internal relapses were common, but whether these resulted from
viscera which remained inert “only to be set free as a persistence of the blood stage infection (i.e. recrudes-
result of some insult, the nature of which is still not cence), or derived from a latent tissue stage was
appreciable to us” [8]. Self-experimentation then pro- unresolved.
vided remarkably accurate descriptions of long latencies
in vivax malaria. In 1900 in London Sir Patrick Manson Early observations on relapse
was investigating the mosquito transmission theory pro- In 1913, Bignami proposed that relapses of malaria all
posed by his protégé Ronald Ross. Upon request derived from persistence of small numbers of parasites
Bignami and Bastianelli kindly provided him with P. in the blood [21]. Although this theory explained the
vivax infected anopheline mosquitoes that had been fed late recurrences of Plasmodium malariae infections in
on malaria patients in the Ospedale Spirito Santo in man, it did not explain several features of P. vivax and
Rome. The mosquitoes were taken to the British P. ovale infections. A much clearer understanding of
Embassy in Rome, and thereafter by the “Indian mail”, relapse in P. vivax malaria was to come from Julius
and duly arrived in London 48 hours later. His son Wagner-Jauregg’s discovery that malaria could cure neu-
Patrick Thorburn Manson, and George Warren (a rosyphilis. Between the 1920s and the 1950s thousands
laboratory technician) both volunteered to be bitten. of patients confined in mental hospitals with neurosy-
Acute attacks of malaria followed after a two-week incu- philis were treated with malaria (malaria therapy). Gen-
bation period, in September 1900. These were treated eral paralysis of the insane was then a uniformly lethal
successfully with quinine [10]. On June 1st the following condition, and at least half the malaria therapy treated
year, after a latent period of nine months, the younger patents were improved and over one fifth were cured. It
Manson experienced a sudden onset of rigors. Relapse was a remarkable period, unique in the history of medi-
of his vivax malaria was confirmed by microscopy [11]. cine, when, as Henry Dale put it, “Man was the experi-
Meanwhile in India, at the end of 1900, Major CF mental animal”. The majority of the malaria therapy
Fearnside infected himself with mosquitoes which had experience was with a relatively small number of para-
fed on vivax malaria patients in the Madras jail. His pri- site “strains” transmitted either by blood passage, or
mary attack of malaria began on January 14th, he suf- more usually by the bites of several infected anopheline
fered a relapse on March 11 1901, and a second relapse mosquitoes. On the 8th September 1922 Professor War-
followed on November 11th apparently without the pos- rington Yorke and JWS MacFie inoculated blood from a
sibility of interim reinfection [12]. These precise patient with simple tertian malaria (P. vivax), which had

malaria therapy with local “strains” [17]. even with recurrence in blood transmitted vivax malaria could be multiple infected anophelines. recorded by Korteweg from 1902 to 1923 (black line) [15-17. been acquired in India.22-29] whereas mosquito infection. but. Importantly it was also noted in The rence in mosquito transmitted vivax malaria could not Netherlands that relapse rates in naturally acquired be [17. by subtraction.90:105Ͳ11 A.atroparvus 20 15 Delayed 10 primary illness 5 Relapseandearly primaryillness 0 J F M A M J J A S O N D Month Figure 1 Long-latency P. Swellengrebel et al showed that malaria transmission usually occurred between the first week of August and the end of October [16]. vivax (and P.20.White Malaria Journal 2011.malariajournal. The Northern Eur- different temporal pattern to those of P. often failed to produce an prevented by curing the blood stage infection.23]. 10:297 Page 3 of 35 http://www. From this it can be deduced that the initial wave of malaria cases derived from inoculations the previous year (pink curve) and. that this was followed by relapses and primary cases with a short incubation period in the late summer and autumn (blue curve) [19]. was required to ensure an acute illness within weeks ment of the primary infection. The most The Dutch malariologists were able to show that their widely used parasite used for malaria therapy in Europe .27] (Figure 3). This “strain” was then used to infect multiple mosquitoes. vivax therefore proved [24-26]. in a further example of courageous patients initially by blood passage. This pointed to an exoerythrocytic stage infections were higher than with mosquito-transmitted of malaria. recur.ovale) malaria followed a to 9 months later [26. The mean monthly number of malaria cases in the village of Wormerveer. they proved that bites by one or transmitted infections. Furthermore whereas [17. Recurrences of vivax malaria commonly unsatisfactory for malaria therapy because blood passage occurred many months after apparently successful treat. It soon became apparent that two infected mosquito were followed by vivax malaria 8 recurrences of P. The Netherlands. vivax in the Netherlands. but its anatomical location remained elusive.19]. and later by mosquito self-experimentation. vivax could have a short incubation per- at the Whittingham Mental Hospital near Liverpool iod if patients were bitten by a large number of infected [22. early febrile illness.com/content/10/1/297 Malariacases Luxemburgeretal TransRSocTropMe 25 1996.22-24]. falciparum opean and Russian “strains” of P. into a patient with neurosyphilis indigenous P.

England [24-26]between 1925 and 1930. investiga- of malaria (any species) before eight weeks was termed tions in India by Sinton and colleagues.21]. and in Florida .White Malaria Journal 2011. was different to that used today. and return of malaria after 24 weeks [8. was a P. eral European centres (Figure 2). The “Madagascar” strain of P. vivax was exten. The vivax relapses had a bimodal pattern with the majority having a long latent period (mode 28 weeks) before the relapse. of indigenous origin. vivax (105 patients) studied by SP James and colleagues at the Horton Hospital. malaria therapy. Epsom. It reliably produced an and Ciuca in Romania with the Madagascar strain of acute infection with high fevers. which was adopted by several upper end of the spectrum of abilities to produce early others. 10:297 Page 4 of 35 http://www.com/content/10/1/297 28weeks 7weeks Figure 2 The temporal pattern of illness recurrence in patients with neurosyphilis artificially infected for malaria therapy with Plasmodium falciparum (87 patients) and the “Madagascar” strain of P. so they probably represented the James’ terminology. These through man and mosquito the Madagascar strain strains were all chosen because of their suitability for apparently became even more virulent (Figure 4) [30]. vivax “strain” isolated from an Indian merchant a recrudescence. was called a recurrence. Swellengrebel in The Netherlands. The European studies. With serial passage malaria therapy in the United States [27-38]. A recurrence infection and relapse. recurrence from 8-24 weeks was seaman whose last port of call had been Madagascar termed relapse. used for approximately eight months later. sometimes relapsed P.malariajournal. and then relapsed again and McCoy strains. The pattern observed by sively investigated in England by James and later in sev. vivax was very similar to patterns of the St Elizabeth again within a few weeks. James in England.

the consequent long illness obscured early nated spontaneously (indicating an effective immune relapses.com/content/10/1/297 Figure 3 Combined results of the preliminary mosquito infection studies of James and Shute in the Horton Hospital. Boyd and Kitchen. England and the self experimentation of Shüffner. not possible [25-31. This acquired immunity and then exhibited the long-latency seen with the suppressed later homologous relapses. our current understanding of P.34. who stu- many weeks duration became a significant confounder.37]. Swellengrebel-de Graaf.34. vivax epidemiology tinction was often unclear as characterization of asymptomatic parasitaemia (and gametocytaemia) latency required reliable long-term follow-up. These single isolate (presumably single or response) or infections in which the primary illness highly related genotype) infections eventually resulted lasted for ≥ 48 days [31. By contrast in solid immunity such that after several episodes of all agreed that if prompt anti-malarial treatment was . died the McCoy strain in Florida extensively.37] (Figure 5). The dis. This confirmed the 8 to 9 month interval from being bitten by one or two infected anopheline mosquitoes and developing vivax malaria by Boyd suggested that some “strains” relapsed after a protracted fever reinfection with the same isolate was few weeks while others had only a primary infection. de Buck and de Moor in The Netherlands as illustrated by Shüffner et al [27]. 10:297 Page 5 of 35 http://www.White Malaria Journal 2011.malariajournal. Swellengrebel. Importantly for Madagascar and St Elizabeth strains [24-30]. and the tended to persist for weeks as disease controlling development of immunity with a febrile illness of immunity was acquired. noted As the object of malaria therapy was a prolonged high that relapse did not follow infections which had termi- fever. Korteweg. Epsom.

The number within the circles refers to the number of patients infected with the “Madagascar strain” at each time and the number over the lines refers to the batch number of the infected mosquitoes. 10:297 Page 6 of 35 http://www. .White Malaria Journal 2011.malariajournal.com/content/10/1/297 Figure 4 The family history between May 1925 and May 1933 of the “Madagascar strain” of Plasmodium vivax as used in malaria therapy at the Horton hospital [30].361 mosquitoes and 1739 patients were infected. Overall 24.

given to terminate the infection then symptomatic patients were generally similar whichever month the relapses of vivax malaria were common. In southern often resulted in an extended incubation period of 7-10 Europe there was often a bimodal pattern with a late months. During the malaria therapy experience it became clear Between the 1920s to the 1940s the long-latency infec- that both the incubation period and the number of tion was regarded as the “usual” P. were to confirm which occurred the year after the falciparum malaria these observations. all pointed clearly to long-latency P.White Malaria Journal 2011. China. The epidemio- more likely was an early infection (incubation period logical studies of malaria epidemics in Sind (now Paki- two weeks). The Dutch had shown first [27].malariajournal. vivax malaria therapy in 375 patients studied at the Florida State Hospital [93]. relapses were determined by the numbers of sporozoites Throughout the endemic areas of Europe vivax malaria inoculated [25-28. In order to ensure that there was a epidemics in these two tropical areas (although other short incubation period preceding the malaria illness interpretations are also possible). the Caucasus. Infections which were allowed to continue until self termination did not relapse subsequently [31]. In contrast the soldiers on both . Italy. Most infections were with the McCoy strain.25. and Greece. Inset is the study of Coatney et al [71] describing 403 mosquito transmitted infections with the St Elizabeth strain of P. During the Second malaria therapy infections were produced typically by World War observations on Allied soldiers fighting in the bites of 5-10 infected mosquitoes. and some were with other local “strains” which he considered to behave similarly. further observations in Japanese occupation forces in Previous theories that seasonal influences were impor. peaked in the late spring and early summer (largely and others later confirmed.pro. This suggested that long-latency P. stan) and Ceylon (Sri Lanka) followed a similar pattern vided that prompt anti-malarial treatment (quinine) was [19.39]. Later clinical and experimental studies.25]. vivax with tant determinants of relapse were largely rejected as the similar illness patterns to the Madagascar and St Eliza- intervals from primary illness to relapse in neurosyphilis beth strains [40-45]. also responsible for the peak of vivax malaria cases reported after the Second World War. vivax was given each time. that low sporozoite inocula from inoculations the previous year) [20. vivax. The median interval to relapse was approximately 9 months. and the more relapses that followed . The more sporozoites that were inoculated. 10:297 Page 7 of 35 http://www. vivax phenotype. and either no North Africa. and treatment or partial suppressive treatment was given.com/content/10/1/297 100 80 60 40 20 0 01020304050 60 ONSET OF SECONDARY ATTACKS IN WEEKS FROM INOCULATION Figure 5 Boyd’s 10 year experience with mosquito transmitted P. infection started [22-26].38]. the summer peak of falciparum malaria [19].

White Malaria Journal 2011. and by then tissue stages of bird malarias blood stage multiplication. the eminent proto. although later pri- Multiple relapses were very common and there was no mate work suggested that relapses might arise from evidence of long latency. vivax to a volunteer recipi. Discovery of the liver stages The term relapse is now used specifically to describe In 1902. intrin- returned to the blood one week later [55-57]. Forty years later Krotoski. It was considered the “typical” P. The existence of unanswered as to how the hypnozoites were woken. and there were well documented. The question remained had been demonstrated unequivocally. parasites only zoites per blood schizont. cence refers to a recurrence of malaria derived from There was therefore no need to postulate a tissue stage persistence of the blood stage infection. Although parasite bodies. vivax hibernans) in which the primary infection .but where was it? SP James. At that time the Plasmodium relapse period even transfusion of as much as 500 mL vivax with infection phenotypes similar to those of the blood could not transmit P. After the Second vivax “strains”. arises after the “awakening” of these hypnozoites and dited as others had tried and failed to reproduce the the subsequent intrahepatic schizogony followed by observations. vivax sporozoite in human malaria went on to divide in cells Today there is a tendency to regard all P.malariajournal. considered sufficiently likely that the Malaria Commis- sion of the League of Nations in 1933 suggested that the Phenotypic variation in P. arrested development of hepatic pre-erythrocytic schi- cal frequent relapse P. Infections occurred vivax in a very heavily infected volunteer who under- at three week intervals if quinine was given. nomic debate over how these “sub-species” should be mental station showed that sporozoites were cleared defined (Figure 6). subsequent relapses originated from an exoerythrocytic although relapse could follow some three weeks later. falciparum recurred -it could always valent in the United States and Southern Europe were be transmitted beforehand by blood transfusion. and. hypnozoites. Shortly afterwards in England (P. Studies conducted over fifty years from the blood within one hour of mosquito feeding on ago indicated that incubation periods. vivax infections clear then that there was a pre-erythrocytic tissue stage [24. A primary illness followed approxi- which preceded the blood stage infection. virulence. and a tissue stage of the malaria life cycle in humans was what determined their remarkable periodicity. zonts [63. vivax together of the endothelial system as did Haemoproteus in birds. Northern Germany. as a single homogenous species.71]. but told the young PCC Garnham to stay in East Africa apparently unusual. keys [59.cynomolgi infected Rhesus mon- relapse rate was very high -in some companies all sol. differed between “strains”. antigenic relationships.com/content/10/1/297 sides fighting in the Indo-Burman and South Pacific definitive studies by Shortt and Garnham identified the campaigns encountered vivax malaria with a very differ.60]. and then in a heroic experiment with P. the eminent British there was often a 7 to 10 month interval before a subse- malariologist. numbers of mero- volunteers. diers were infected and all relapsed. finally acquired the infection in New Guinea [50-52]. working with son strain” isolated from a soldier of that name who had Garnham and colleagues at Imperial College. Sometimes the latency could be as long exoerythrocytic stage in the primate malarias that he as one year. stage . The relapse of malaria. and also that mately two weeks after mosquito inoculation. which are probably quinine. identified the dormant stages or “hypnozoites” of P. was so convinced that there must be an quent relapse.64]. 10:297 Page 8 of 35 http://www. Several sic drug susceptibility. Relapses were frequent and the as the liver. did not identify the persistent stage. In volun. recurrences of malaria derived from persistent liver zoologist Fritz Schaudinn reported that he had observed stages of the parasite (hypnozoites) whereas recrudes- direct infection of erythrocytes by sporozoites [53]. “Madagascar” and “St Elizabeth” strains which were pre- ent.25. By the 1930s this theory was largely discre. Finland and Central cystis (then Plasmodium) kochi in the hepatocytes of Russia the long incubation phenotypes were prevalent African monkeys [58]. and seven went open liver biopsy [61-63]. cases reported of latencies greater until he had found it -and so he did! In 1947. whereas if P.34.gallinaceum took place in substantial phenotypic variation between Plasmodium the brain capillaries of chickens [54]. and relapse intervals all researchers had noted earlier that in the latent or inter. Garnham than two years. This classic work still week intervals following mepacrine treatment [42-49]. site of pre-erythrocytic development in primate malarias ent relapse pattern. Fairley’s brilliant work at the Cairns experi. The “type strain” for this tropi. Further north in the Netherlands. first in P. There has been corresponding taxo- World War.31. vivax phenotype was the “Ches. three years before his death. identified the pre-erythrocytic development of Hepato.cyno- teers infected with the Chesson strain 80% of relapses molgi and P. have since been demonstrated in liver cell cultures [70] remarkably little is known of their biology. Scandinavia. vivax responsible for relapses in the liver occurred within 30 days of initial treatment with [65-69]. but the human malaria In 1937 James and Tate showed that the exoerythrocytic therapy and volunteer studies showed that there was development of Plasmodium. vivax. and in the case of P.

These observations were similar to those in was given for treatment [17. The administra- explanations for relapses which emerge two to eight tion of an effective schizontocidal drug allowed better months after sporozoite inoculation. notype vivax malaria [75]. vivax phenotypes and the usual patterns of primary illness and relapse (after Bray and Garnham [69]). definition of relapse periodicity than the malaria therapy There was one very important and puzzling feature of studies (where the objective had been to produce a sus- the long-latency P. occurred 8 to 10 months after inoculation Thus after the long latent period the subsequent inter- [17-20. The thickness of the lines gives a rough approximation of proportions. with intermediate relapse characteristics have been Following the Second World War artificial infection reported but not well described from sub-tropical areas. there may be other ferent anti-malarial treatment regimens.19.malariajournal.White Malaria Journal 2011.27-29.23.76]. vivax infections which still requires tained high fever). In contrast infections from In artificial infection studies latency was independent of some parts of Russia were documented which had a sec- season [22-26. the seminal chemotherapy studies of Sinton and . 10:297 Page 9 of 35 http://www. intervals in the tropical frequent relapse “Chesson” phe- tude (and shorter summer mosquito breeding seasons). vivax treated in soldiers fighting in the Indo-Bur- periodicities.24. In the more prevalent tropical strains explanation in any theory which seeks to explain relapse of P. the relapses were documented to occur at usually occur with intervals of approximately 3 to 4 intervals of 3 to 4 weeks (as in the Chesson strain) weeks following quinine -or 6 to 8 weeks if chloroquine [42-52.72-74]. studies were conducted in several locations to study dif- although as will be explained later. In the past twenty years infections ond long-latency after the first relapse(s).43. and the inter-relapse (circa two weeks) declined steadily with increasing lati. It seemed that the propor.com/content/10/1/297 Figure 6 Diagram of the different P.23. once the relapse had occurred (after a mese and South-West Pacific campaigns in the Second latency of 7-10 months) subsequent relapses would then World War.71]. vals were similar to the incubation period in temperate tion of infections which had a short incubation period infections with early relapses.71-73] (Figure 7).

Note the frequent relapse pattern after a long interval with Korean vivax malaria. not occur for nine or 10 months. “normal” incubation period of two weeks.81].71-73]. . Interest in the long-latency 1968 [28]. In long term observations of infections mates. when increasing sporozoite doses of the tropical “Ches- would return to clinics for many years complaining of son” strain were inoculated the incubation period shor- recurrences of malaria.com/content/10/1/297 Figure 7 Schematic diagram by Hankey et al [79]of relapse patterns following Korean vivax malaria (upper panel) and tropical frequent relapse P. 10:297 Page 10 of 35 http://www. If ≥ 1000 blem in soldiers fighting in the Korean war [79]. This. or longer. When the inoculum of sporozoites was small phenotype revived during the early 1950s as P. vivax (lower panel). These terminal long intervals did not appear to have a vations were made with a North Korean strain used for fixed periodicity (Figure 8). It was noted that even with a single infected with the St Elizabeth strain there was a clear bimodal mosquito bite some Chesson strain P. colleagues in Kasauli. Similar obser. During sporozoites were inoculated illness occurred after a this period patients who had received very heavy inocu. vivax infections pattern in which a long pre-patent period (~300 days) relapsed after intervals which were as long as a year only occurred following smaller sporozoite inocula after a series of regular “short interval” relapses [75].78]. (Figure 6) Several other For long-latency vivax malaria from Northern climes the important observations were made in the artificial infec- sporozoite dose determined the clinical phenotype tion studies conducted in humans and experimental pri- [27-29. and a series of experimental inves- relapses in others despite anti-malarial treatment gave tigations in chimpanzees [80. (more reflective of natural infection) [71].White Malaria Journal 2011. The long latencies and the long tened and there was no evidence for a long prepatent relapse intervals in some infections and the multiple period [75.77. led Garnham to pro- rise to the old saw that “you never got rid of malaria”. typically soldiers from the Second World War.80].38. Himachal Pradesh. vivax (10-100 sporozoites) the initial parasitaemic illness did malaria which had a long-latency was an important pro.69].malariajournal. pose that the ratio of hypnozoites to immediately developing forms in the Korean P. By contrast lations. vivax strain was Relapse intervals 999:1 compared with the Chesson strain where he esti- Effects of sporozoite inoculum mated the ratio as 50:50 [68. India in the malaria therapy in a Moscow hospital from 1953 to 1920s and 1930s [32.

vivax studied by Coatney et al [75]. Some were rechallenged as indicated. Thus intervals .82. and also P. For example if ten important consideration for concomitant activation of genetically identical hypnozoites are activated the hypnozoites with different genotypes in endemic areas relapse interval will in 91% of occasions be shorter than as will be discussed later.com/content/10/1/297 397days Figure 8 Relapse patterns in volunteers in the USA who were infected by a single mosquito bite with the Chesson strain of P.numerical probabilities and immunity. This is because there is genotypically distinct hypnozoites may be activated but.cynomolgi activated and most rapidly multiplying parasite that in Rhesus monkeys. This is an parasites cause patent infection. identical organisms and it is the progeny of the earliest son strain P. relationship is also clearly seen in Schmidt’s studies of sured until the progeny of the most rapidly growing P. 10:297 Page 11 of 35 http://www. Two factors are [71]. The the more hypnozoites that are activated the shorter is simultaneous activation of several hypnozoites will the average interval between relapses (Figure 11). In contrast the initial inter-relapse intervals of Ches. natural phenotypic variation even amongst genetically on many occasions. only one or two genotypes will be .White Malaria Journal 2011. The interval from inoculation to relapse (~9 likely to have explained these gradually lengthening months later) shortened with increasing inocula.cynomolgi in monkeys [82] (Figure 9). vivax in volunteers. This shorten the relapse interval because the interval is mea.52. were remarkably regular although become patent first. This is best illustrated in the studies they gradually lengthened with each successive relapse of Coatney et al with the long-latency St Elizabeth strain [51.malariajournal.75. In natural settings multiple if one hypnozoite is activated.83] (Figures 9 and 10).

and treatment such that they never reach size of sporozoite inoculum there was a corresponding patency. whereas with increasing response. In Schmidt’s primate studies genotype. 10:297 Page 12 of 35 http://www. shortening of the interval. but this did not affect the growth may be suppressed by fever. with 5 × 106. or asexual there was an early infection. The other hyp. was clear evidence that late relapses were attenuated if nozoites’ progeny may reach patency later. In the studies of the St Elizabeth strain there (Figure 9) the lengthening of inter-relapse intervals was . interval . respectively.White Malaria Journal 2011. 5 × 102 and 5 sporozoites. These observations suggest Effects of immunity that inoculum size is more important than immunity in The second factor accounting for lengthening inter. Monkeys in groups A. The infections were induced with different numbers of injected sporozoites as indicated and treated repetitively with chloroquine. B. Some monkeys were rechallenged and some were finally given radical treatment with primaquine in addition.com/content/10/1/297 Figure 9 Relapse patterns of P. C.malariajournal. 5 × 104. illness.cynomolgi infections in Rhesus monkeys studied by Schmidt [82]. detected subsequently at clinical relapse. and D were inoculated.at least for the first relapses with genetically tion of blood stage immunity against the single infecting homologous parasites [71]. immune interval to latency (Figure 11). determining the duration of latency or the inter relapse relapse intervals in artificial infections was the acquisi.

but were uncommon in Cauca- 16 days [83]. then relapse did not contrasts with the natural setting where anopheline occur and reinfection with the homologous strain was mosquitoes display a wide range of infectivities depend- not possible [34]. It Figure 10 Lengthening intervals between sequential relapses was noted that haemolytic reactions occurred sporadi- in individual volunteers who were infected with the Chesson cally with plasmoquine in patients of Asian. was a consistent salivary gland sporozoite loads in sibling mosquitoes. quitoes selected for maximal infectivity based on which persists for many months.82. vivax [34]. which also has a term- 45 inal half-life of over one month.16d)only half-life of over one month. and their synergistic action with quinine.White Malaria Journal 2011. but a different elimina- tion profile to mepacrine. given early (on the second day of patency). Artificial infections provided invaluable information but teers infected with the Chesson strain of P.e. vivax invasion of erythro- animals received chloroquine treatment which was cytes in this population. Boyd noted that if the initial infection was mental studies to coincide with maximum infectivity allowed to run its natural course. This was explained later by the epidemiology of glucose-6-phosphate dehy- drogenase deficiency. Blood apy followed the bites of 5-10 infected anopheline mos- stage immunity against homologous strains of P.52. In the Southern United States it less prominent than in the human investigations proved difficult of impossible to infect patents or volun- [51.84]. from three to seven weeks) [40.47]. Later chloroquine. observation in malaria therapy and challenge studies The timing of inoculation in malaria therapy and experi- [34. were demonstrated within years of their introduction in the mid 1920s [85-87]. vivax relapse. and reduces parasite densities.83] and increasing inter-relapse intervals teers of West African descent with P. Diamonds represent median values. each of whom was treated with quinine for South European descent.47]. and consequently reduced their frequency.71. a drug which has a terminal elimination Quinine(2g/d. sians originating further north [88].malariajournal. vivax [75]. The artificial infections together with slower asexual growth rates resulting in the majority of volunteer studies and in malaria ther- from the acquisition of asexual stage immunity. and all blood group receptor for P. Larger 40 doses of the anti-malarials resulted in longer intervals to relapse consistent with a concentration-dependent slow- ing of asexual growth rates. Slowly eliminated anti-malar- 35 ials delayed the onset of P. it was observed that early relapses were delayed by approximately thirty days com- 50 pared with quinine treatment (i. African or strain of P. The relapse 25 preventing properties of these drugs. quina- 55 crine) in 1932. although in was later shown to reflect the absence of the Duffy these experiments the inocula were very large. The infections were in non-immune series of short intervals. but importantly 30 these drugs did not appear to reduce the overall number of relapses experienced [47] (Figure 12). they differed from natural infections in several impor- that occasionally a very long interval would follow a tant respects [89]. Only the 8-ami- noquinolines reduced or prevented relapses. which results in reversion to the mean broad range of local parasites which controls symptoms intervals associated with single hypnozoite activation. was found to delay early relapse appearance by two to six weeks [40. vivax. as Coatney had done earlier in volun.com/content/10/1/297 Effects of drugs Numberofdaysbetweenrelapses Since the introduction of mepacrine (atebrine. 10:297 Page 13 of 35 http://www. areas have usually developed significant immunity to a cessive relapses. Adults in the malaria endemic from both the “running out of hypnozoites” with suc. vivax. Schmidt also observed in the Rhesus monkeys infected with P. The inocula in . Contrasting artificial with natural infections cynomolgi [82]. ing on sporozoite age and other factors. This were seen only after 12 or so relapses [82]. 20 Two other key observations were made during this 1234 1 2 3 4 early era of malaria therapy and drug evaluation which Numberofrelapses were not satisfactorily explained until decades later. Thus the steadily increasing adults whereas the burden of vivax disease in endemic intervals between the homologous strain relapses result areas was in children.

vivax epide- 80 miology resulting from low sporozoite inocula in areas Quinine of low seasonal transmission. g Relapse (%) artificial infections were therefore usually “supranormal”.5 .2 g/day for 4-7 days (N = 82). If Garnham’s estimates (50:50 ratio of immediately developing parasites to hypnozoites) are 40 correct this corresponds to a median of 5 hypnozoites in tropical P.com/content/10/1/297 Dayoflaterelapseonset 300 290 280 270 260 250 0204060 0204060 Daysofpatentparasitaemia Infectiveinocula Figure 11 In infections of volunteers with the St Elizabeth strain of P.4 g/day for 7 related group of genotypes.malariajournal. 2 g/day for 14 days (N = 75). vivax infections. In contrast multiple unre- days (N = 69). .White Malaria Journal 2011. Even if 0 20 40 60 80 100 120 140 160 Days after completion of treatment these infections originated as a mixed genotype infec- Figure 12 Cumulative proportions of relapses in soldiers with tions in the donor patient it is likely that with multiple vivax malaria acquired in the Pacific and treated subsequently passage in malaria therapy the “strains” became purified with different anti-malarial drug regimens in Chicago. there was no evident relationship between the occurrence or duration of the primary illness and the long-latency interval before illness (left panel). 10:297 Page 14 of 35 http://www. Regimens through successive interbreeding to a very closely were quinine. and lated genotype infections are common in natural quinine 2 g/day and plasmoquine 60 mg/day for 14 days (N = 72) [47]. whereas there was an inverse relationship between sporozoite inocula (assessed semi-quantitatively from sporozoite numbers in the salivary glands and number of infectious bites) and the latency interval. The “strains” of P. Median sporozoite inocula Mepacrine Chloroquine in natural infections are estimated to be less than 10 60 sporozoites [90-92]. vivax 20 used in malaria therapy were likely to have been of a Quinine+Plasmoquine single (albeit evolving) genotype or very closely related 0 interbreeding genotypes which were passaged through a very large number of patients over many years. infections. mepacrine 0. chloroquine 1. vivax [71]. 100 This resulted in reliable infections but did not bring out the important stochastic component of P.

This high Without radical treatment the proportion of patients mortality reflects the underlying condition.51. illness. In total. North Africa.25% of patients have four or more “strains”. Korea. India. atebrine) was stopped P.000 Geographic distribution patients with acute vivax malaria have been reported in Overall there was good evidence for the presence of the the available medical literature in English (> 300 publi. endemic areas could not exclude reinfection. then relapse becomes the predominant cause of single genotype depended on the duration of preceding vivax malaria illness. the proportion of these was undoubtedly contributed to by the infection as well. In other words the relationship between endemic areas but in malaria therapy this was underta. Overall the mortality asso. more relapses. vivax endemic areas of the world).84. although it who experience one relapse. 9% had 10 or more relapses [76] distributions (Figure 17). Madagascar. It is units all soldiers had relapses). North and Central America (Figure 17). South East Asia and the island of New Guinea (which sure. vivax infections which relapse is ber of relapses is. The vivax phenotypes pertains today.41.com/content/10/1/297 The malaria therapy patients usually had neurosyphilis enforced in soldiers it became clear that almost all and were often very frail. This is function of the sporozoite inoculum and immunity (if important when considering radical curative drug effi- any). The reported relapse rates varied from 0 Long incubation period P. South East Asia and Oceania.24. and in volunteer studies was performed to demonstrate the “strain specificity” of the immune Thus if “x” is the fraction of patients experiencing one or response.42. In the Second World War 70 to 80% of soldiers has much higher levels of malaria transmission than fighting in the South Pacific had relapses (and in many most other P. regarded as the mildest human malaria) [17].malariajournal. the ure 14). Where both are present (Figure 15). then the fraction experiencing “n” or more relapses is approximately xn The proportion of infections which relapse A plot of the logarithm of the proportions versus num- The proportion of P. the treatment responses in over 87. vivax malaria therapy was approximately the mepacrine (quinacrine. horn of Africa. 10:297 Page 15 of 35 http://www. the Middle East. On the other hand studies conducted in not been eradicated this geographic distribution of P. In Hill and Amatuzio’s also evident that both phenotypes overlap in geographic series of 222 patients. Frequent relapse “strains” were reported in parts recent studies. and the proportion The objective of treatment in natural infections is cure. and in of South America. fifty years old. Southern Russia. cient. although the presence of a Second World War [94]. Two-thirds of the reports are over China.000 relapsed (Figure 13 and Fig. malaria could be prevented. three or more etc relapses is exponential.76. patients who have a second relapse. is constant (Table but in malaria therapy quinine was used to “damp 1). long-latency “Madagascar” relapse phenotypes in Eur- cations) of whom ~17. culation was usually unsuccessful [34. Reinfection with a different genotype was usual in 52. two or more. of these who have a third and so on. Central parts of and military reports. them. although there is very relapse rate was very high in soldiers who were generally little contemporary information apart from reports from non-immune and presumably experienced intense expo. but that several weeks after ciated with P.47-49. not to eliminate gical or experimental setting [23. ken only occasionally if the first infection was insuffi.93] (Figure 5). were often two months or less. Pakistan and Northern India.malariae infections -generally vivax relapses started to occur [47]. Relapses were estimated to account for together it would be very easy for the long-latency infec- about half of all vivax malaria hospitalizations in the tions to go unrecognized. Central Asia. from ≥ 1 hypnozoite) (text box on pharmacodynamics). Afghani- malarial treatment regimens. den of hypnozoites (because each relapse must derive termination the relapse did not usually occur and reino. ope.85].White Malaria Journal 2011.79.28. It follows that once symptomatic relapse rates exceed In this setting the probability of relapse with a presumed 50%.29. is heavily biased to adults stan. As described earlier if artificial sporozoite induced cacy as it provides an indication of the minimum bur- infections were allowed to continue for weeks until self. vivax (”hibernans“) was pre- to 100%! Many relapse rates are likely to have been valent in Northern Europe and more Northern parts of underestimated as follow-up periods. 7% (and was 10% with P. Tropical “strains” relapsed more than temperate approximately 6. therefore.72. Thus. particularly in Russia. This experience of a wide variety of anti. for areas with 50% relapse rates region. linear with slopes varying often thought of as an intrinsic property of the malaria depending on the geographical and epidemiological set- parasites which varies considerably by geographic ting (Figure 16). Once chemoprophylaxis was spring vivax malaria peak while vector densities are still . This appears to obtain in each different epidemiolo- down” the more severe infections. But the relapse proportion is also clearly a relapses per incident symptomatic infection. the majority of studies radical treatments were being It is highly likely that in those areas where malaria has evaluated. the numbers of patients who experience one or more.

Eliza- unrecognized outside the sub-continent. which is similar to other types. Reinfection despite its obvious importance.1% (Delhi) [97-99]. was considered unlikely. from the Delhi area [98] where the authors concluded bal burden of vivax malaria today is the tropical fre. This ment of between 8. because India harbours the major. The greatest uncer. Group have recorded relapse rates following chloroquine treat. Indian sub-continent. beth-type strain that has a long period of latency Prospective studies from India over the past 25 years between the primary attack and the first relapse. vivax relapse rates without radical treatment in published studies conducted between 1920 and 2010. It is generally thought. vivax in the world and clearly has both pheno. if it exists. The horizontal scale represents the study location from West (United States) to East (Pacific).malariajournal.96]. This remarkable gap in our understanding of the Southeast Asian strains such as those from Thailand global epidemiology of vivax malaria seems to have gone and Vietnam. has not been well charac- desh) and 40. intermediate group. “Based on the foregoing epidemiologic features.9% (Madhya Pra. There chloroquine only and who were followed for one year has been very little recent interest in this question had a relapse (17 within six months) [99]. The ratio of short to long-latency relapse phe- 150 patients with vivax malaria and treated with notypes in Aligarh. Group I is the tainty is the epidemiology of relapse phenotypes in the tropical or Chesson strain type of frequent relapsing P. attack and the first relapse. vivax with a short period of latency between the primary ity of P. that the majority of the glo. low would be an epidemiological pointer [19.White Malaria Journal 2011. Group III is the temperate or St. Uttar Pradesh was estimated as 4:1 . distinct relapse patterns were observed in the present vivax to be sequenced fully was a long-latency pheno. A very wide range of treatments were used in a very diverse range of patient groups. three quent relapse type (although interestingly the first P. Each circle represents one study arm. tion in northern India is polymorphic. II is intermediate between these two types” [98]. study.20]. and it can be concluded that the P vivax popula- type from El Salvador-Sal 1) [95.620 Relapse(%) 100 90 80 70 60 50 40 30 20 10 0 0 50 100 150 200 Study West East Figure 13 P.6% (Orissa) and 8.com/content/10/1/297 Studiesofvivaxmalariatreatmentwithout8Ͳaminoquinolines Studieswhichenrolled:5Ͳ 5528patients N=31. 10:297 Page 16 of 35 http://www. In Mumbai 19 out of terized. Higher rates were reported but is by no means certain.

tioned in India in the 1920s and 1930s. which may be more 143) were symptomatic.malariajournal. vivax occurred in one patient Israel. Silachamroon et al studied effective against the long incubation or long-latency adult patients in Thailand (infections from Burma. 10:297 Page 17 of 35 http://www. Yorke. Reappearance of P. recently Luxemburger et al studied 342 children with cial infections of Indian origin [12. 63. Turkish immigrants to Germany. phenotypes. and US soldiers on day 21 and in 8 by day 28. and this is consis. although studies in Eritrean immigrants to [106]. vivax relapse rates following treatments which included 8-aminoquinolines (mainly plasmoquine or primaquine).060 100 90 80 70 60 50 40 30 20 10 0 0 20 40 60 80 100 Study West East Figure 14 P. More observations of Fearnside. Most reappearances of parasitaemia (85%. Overall relapse rates from India have been rela. 121/ malaria in temperate countries. and in prisoners of war in Thailand [46].109] and also the early Burma. .22. and MacFie with artifi. and was tent with evidence in the past from Northern India. There are few acute vivax malaria treated with chloroquine on the data on temporal patterns of relapse in travellers in north-western border of Thailand in 1995 and 1996 recent years.com/content/10/1/297 Studiesofvivaxmalariatreatmentwith 8Ͳaminoquinolines Relapse(%) Studieswhichenrolled:5Ͳ 13. observed in soldiers fighting in North-East India and Pakistan and Afghanistan [108. Data from travellers returning from the Indian sub-con. giving a 28-day cure rate returning from Somalia suggest the presence of long. the relapse/re-infection rate was 63% (95% CI 57- Most travellers receive radical treatment for vivax 69%).720patients N=56. infections than against the tropical frequent relapse tively low by comparison with South-East Asia [97-107]. By day latency phenotypes in the countries of origin [110-112]. Otherwise as for Figure 13 [100]. The tropical frequent relapse phenotype was docu- tinent have suggested that long incubation-period P. of 97% [95% confidence interval (CI) 95-99%].White Malaria Journal 2011. mented by Sinton and colleagues in British soldiers sta- vivax may be common in the Punjab.23].

com/content/10/1/297 Figure 15 Proportions of P. or Cambodia) with acute vivax malaria who relapses emerged from the liver before the eighth day were treated with either 5 days (N = 157) or 7 days (N after starting anti-malarial treatment. Thailand. The artemether-lumefantrine treatment was 38% (the total timing of the relapses suggested that very few if any number may well have been higher because of later Table 1 Relationship between the proportion of patients relapsing with vivax malaria and total number of relapses experienced Proportion of incident P.3 60 1.2% and 47. vivax infections followed by ≥1 relapse (%) Mean number of relapses per incident infection 90 8. In Papua Indone- = 159) of artesunate monotherapy [107].3 80 4.5 50 1. vivax relapses in 222 US servicemen who had fought in the South Pacific in the Second World War [76].43 20 0.0 70 2.11 .White Malaria Journal 2011. 10:297 Page 18 of 35 http://www.malariajournal. Relapse rates sia the relapse rate estimated at six weeks following within 28 days were 52.0 40 0.67 30 0.25 10 0.8% respectively.

vivax relapse phenotypes in Brazil [115. volunteers infected with the Chesson strain in the United States [75] and children followed prospectively in an evaluation of an ineffective malaria vaccine (SPf66) in northwestern Thailand [157]. US soldiers with vivax malaria acquired in the Mediterranean area (two series)[45. In many areas adults are more likely to present to malaria clinics than children. Inset shows proportions on a log scale and numbers of patients studied.malariajournal. patients receiving malaria therapy with the McCoy strain in the United States [93]. In India the The effects of age peak age of malaria presentation is often in young adults In malaria endemic areas. vivax relapses taken from eight different clinical series: These were US soldiers with vivax malaria acquired in the South Pacific (2 series) [49.76].Pacific N=563 Malariatherapy. endemic areas. the age profile of P. a recent report from Rio ence. and another from decline with age.White Malaria Journal 2011.Mediterranean N=155 60% Children. British patients receiving malaria therapy with the Madagascar strain [24. suggest age is a significant confounder in epidemiological assess- that long-latency forms may coexist with frequent ments based on passive case reporting in many P.116]. In French Guiana the relapse suggests much more rapid acquisition of immunity than rate in children was 70% (nearly all relapses occurred for P. vivax is generally regarded as “tropical fre.Italy N=540 10 Soldiers. It is likely that immunity and therefore Brasilia describing long-latency in three patients. matic relapses per mosquito sporozoite inoculation iod of between three to 12 months. emerging relapses) [113]. falciparum [117] (Figure 18). It also suggests that relapses are probably partially de Janeiro that six of 80 travellers presenting with vivax suppressed in older patients. Yet in border of Thailand. dies suggested similar transmission rates (at least in fection could not be excluded [114].Madagascarstrain N=161 70% 20 Malariatherapy.Thailand N=602 5 50% 2 40% 1 0 3 6 9 12 15 30% Relapses 20% 10% 0% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Relapses Figure 16 The proportions of patients experiencing successive P. 10:297 Page 19 of 35 http://www. patients receiving malaria therapy with a local “strain” in Moscow [28]. Thus both the proportion malaria (who had returned from the Amazon region and of infections which relapse and the number of sympto- not received chemoprophylaxis) had an incubation per.Greece: N=180 Soldiers.Chessonstrain N=18 100 Malariatherapy.47]and Italy[156]. Although South terms of measured entomological inoculation rates) so it American P.Moscow N=64 80% 50 Soldiers. Entomological stu- within three months) but both recrudescence and rein. German soldiers who acquired vivax malaria in Greece [41]. vivax malaria the indigenous population living in transmission areas a .com/content/10/1/297 Proportion(%) Proportion(%) Soldiers.McCoystrain N=149 Soldiers. is likely that relapse contributes to much of this differ- quent relapsing” in phenotype.25].SouthPacific: N=222 90% Volunteers. such as the north-western (and often with a predominance of young males).

separates. The 8-aminoqui- noline plasmoquine (plasmochin. age incidence profiles [106]. and without genotyping it may be difficult or impossible to distinguish the two phenotypes within an endemic area (Figure 22). Areas where tropical “frequent relapse phenotypes” are prevalent are shown in pink. Early relapses of P. This applies to bute to the low apparent relapse rates reported from the both falciparum and vivax malaria when malaria is Indian sub-continent. It seems likely then that malaria clinic data are not necessarily representative of disease epidemiology in some endemic areas.falciparum Drug effects on relapse Before the Second World War most malaria infections 600 were treated with quinine. and that studies of Incidence/1000personyears Luxemburgeretal children living in the endemic areas of the Indian sub- TransRSocTropMedH 1000 1996. Usually in endemic areas it is chil. Areas where both frequent relapse and long-latency phenotypes have been reported are shown in purple.vivax P.vivax Frequentrelapse+longlatencyP.vivax FrequentrelapseP. but with increasing control falciparum (by both sexes) by early adulthood which reduces the declines more rapidly than vivax. significant degree of immunity should have been gained uncontrolled. Sinton and colleagues in India soon provided 0 evidence that plasmoquine synergized with quinine in 0 5 10 15 20 25 30 35 40 >40 the treatment of acute vivax malaria and also reduced Years the rate of recurrence (mainly relapse) [85. vivax were observed to occur approximately three to four 400 weeks after starting quinine treatment. replaced the two-month regimens previously in vogue . The paucity of data from children may contri- dren who bear the brunt of malaria.vivax? Figure 17 Approximate historical distribution of P. there is evidence that long-latency phenotypes are present in both areas (particularly across the North of India). 10:297 Page 20 of 35 http://www.White Malaria Journal 2011. pamaquine) was evalu- 200 ated clinically shortly after its discovery in 1924 in Germany. Although both South America and India are generally considered to harbour frequent relapse phenotypes predominantly.com/content/10/1/297 LonglatencyP. and areas where long-latency phenotypes were prevalent are shown in grey.malariajournal.86].90:105Ͳ11 continent might reveal higher relapse rates. “Sinton’s Figure 18 The epidemiology of vivax and falciparum malaria in regimen” of one week’s quinine plus plasmoquine was an area of low seasonal transmission on the Western border of endorsed by the League of Nations and generally Thailand. 800 P. and their epidemiology number of relapses. vivax latency phenotypes.

When mepacrine was developed severe haemolysis. but were relapse (radical cure) [83-88]. usually then presented six to eight weeks quinoline development programme also uncovered the after the primary infection rather than three weeks later. They considered the risk exceeded the benefit [121]. significantly. Mepacrine treat. mon genetic defect of humans [128. vivax [125. USSR where the positional isomer quinocide was pre- quine which resulted in increased plasmoquine concen. The British were more willing to use plasmoquine than the US forces who dis- continued its use during the war. Exposure was predominantly in 1950.129]. and was well documented in all the tropical thea- tres of war. all soldiers were given treatment had suppressed the expansion of the blood a radical curative regimen of 15 mg base/day for two stage infection (post treatment prophylaxis). vivax [47]. The 8-aminoquinolines also have significant trations and oxidative toxicity. Mepacrine was very slowly knowlesi) although resistance in the blood stage infec- eliminated and extensively distributed [119]. Immediately after the Second World War the enormous research effort to find new anti-malarial drugs gave chloroquine (discovered in 1934 and initially overlooked). used chloroquine-primaquine regimen should therefore ment markedly delayed the early relapse of tropical P.White Malaria Journal 2011. they were not prevented [47] relapses was observed in US soldiers returning in 1950 (Figure 12).120]. In the 1930s the newly introduced sulphonamides tolerated in the dose regimens necessary to prevent were shown to have activity against malaria. were both shown to have pre-erythrocytic activity but not radical Figure 19 Numbers of US servicemen admitted to hospital in curative activity. This proved very unclear whether the relapse seen later was the first effective. reason why some patients of African or Asian origin as was usual following quinine. and later pyrimethamine. but recom- weeks. atebrine) in primaquine were developed and evaluated between 1944 1932 and its subsequent introduction provided a sim. whereas Caucasian patients used as a prophylactic it suppressed infections for at originating from Northern Europe did not haemolyse least one month after stopping the drug [47. It was an tion can be induced experimentally [127]. Proguanil. vivax (and Plasmodium drugs simultaneously [118].malariajournal. it acted only on the blood stage para- sites. Relapse of vivax malaria was a major problem in sol- diers.119. It was weeks during their return by sea [125]. and precluded using the blood stage activity against P. 10:297 Page 21 of 35 http://www. Mepacrine NumberofP.126. X-linked Glucose-6-phosphate dehydrogen- Thereafter relapses followed soon afterwards in tropical ase (G6PD) deficiency was discovered as the most com- areas where frequent relapse “strains” were prevalent. better tolerated 8-aminoquinolines pentaquine and then The discovery of mepacrine (quinacrine. proguanil. Furthermore resistance arose readily to the USA each week with vivax malaria following their return the antifol anti-malarials in P. a result of studies on the relatively drug-sensitive Kor- Extended follow-up studies suggested that although ean P. The more effective and more effective against P. The Chesson strain had been shown to be relapse which had been suppressed for three or more more “resistant” to 8-aminoquinolines [124]. and new more effective and less Exposure toxic 8-aminoquinolines. ferred).com/content/10/1/297 [16].vivaxrelapses (atebrine. be considered a combination treatment. The dose of and many months later where long-latency “strains” primaquine recommended globally was chosen largely as were prevalent. After a very high rate of the relapses were delayed. The 8-amino- vivax which. somewhat better tolerated. It was assumed that the slowly eliminated from the Korean war (Figure 19). Chloroquine was clearly the best anti-malarial yet to be discovered. treatment although dard radical treatment of vivax malaria (except in the there was a pharmacokinetic interaction with plasmo. falciparum than P. and 1955 [122-126]. or whether the first relapse had been prevented mendations for a higher dose of primaquine (adult dose: altogether and it was in fact the second relapse (but the first clinical evidence of relapse) that became patent six to eight weeks after starting initial treatment. from the Korean war.130]. Figures taken from the Office of the US Surgeon General. The widely effective treatment of vivax malaria. quinacrine) was the main drug used by all sides in the Second World War. Although plasmo. quine was moderately effective it was relatively poorly . possibly because reac- tions were common in African-American soldiers (pre- sumably G6PD deficient individuals who haemolysed). but like quinine and mepacrine. Primaquine took over as the stan- pler. vivax.

was it pharmacokinetic or pharmacody. related compounds has not been explored further.5 mg/base/kg for 14 days) (Table 2). In Ethiopia where early relapse rates in vivax namic?). If primaquine at a dose of 0. In East Asia and Oceania a similar ducted a formal interaction study which provided pattern pertains today. In the 1950s. and therefore should be curative against nent. weeks at Kasauli (above the level of malaria transmission moquine) was more effective in prevention of relapse at an altitude of 6.White Malaria Journal 2011. Baird has recently pointed out that the malaria infections are highly likely to be relapses. standing of the epidemiology of relapse. Sinton’s work in India had the Indian quinine and malaria inquiry under the newly suggested that quinine and plasmoquine were synergistic formed Central Malaria Bureau. and there is no difference in susceptibility between the groups. ogy evidence on these important points is needed. vivax synergy (i.e. 0. and whether it extends to other quinolines or malaria are much lower.e. then it requires higher dose (i. More clinical pharmacol- tropical and temperate phenotypes and a better under. the with food was not emphasized sufficiently at this time probability of vivax recurrence after falciparum malaria and thus later dosage recommendations may have been is also age related [138]. and this effect is consistent across all patients then the post treatment number of VAH is group A = 39 or 40 group B = 2 or 3. [136-138].e. this proportion is also lower. As the treatments given for falciparum malaria are 75 mg base) were recommended in the Indian subconti. chloroquine and primaquine [132].e. much improved tolerability of primaquine when taken Furthermore. these recurrent [97-105.000 feet in Himachal Pradesh). Five day regimens of primaquine (total adult dose 20). Between March 1924 in the prevention of relapse in vivax malaria [85. as with relapses after vivax malaria. In hindsight there was no very good reason for this. The reason for this falciparum are followed by an infection with P. because the tropical phe- are equivalent but have higher burdens of hypnozoites notype is usually associated with a greater number of which could be activated immediately and so require a relapses than the temperate phenotype. 7% [139]. although there was no evidence they were effective the blood stage infections of P. It is likely that the distribution of VAH is random among the patients and therefore conforms to a Poisson distribution. Ruhe et al ish soldiers. vivax infection are very similar to the intervals mg base/day primaquine regimen usually given for 14 between acute vivax malaria and the first relapse (Figure days. Of 76 with the St Elizabeth strain than sequential administra. As noted earlier. These numbers represent the minimum number of viable activatable hypnozoites (VAH) i. 1921 Major JA Sinton VC was put in charge of is the question of synergy. limited by perceived or observed poor tolerability [134]. then the total number of relapses/100 patients is group A = 395. and In the recent reawakening of interest in malaria it has it might have been better to recommend higher doses been suggested that resistance to the radical curative for all frequent relapse parasites (i. Several lines of evidence Table 2 Radical treatment pharmacodynamics. in South-East activity of primaquine has emerged [135] . falciparum infection and the subsequent vivax malaria. Alving and colleagues con. In East Asia a remarkably high evidence of marked synergy between both quinine and proportion (20-50%) of symptomatic infections with P. vivax. The intervals between the onset of treatment The mid 1950s saw a decline in clinical research on for the acute P. . soldiers completing follow-up 30 (39%) had subsequent tion [131]. group B = 24. vivax malaria [77]. Thus we would expect 13 to 20 times more relapses in group A compared to group B. there are 16 times more in group A compared to group B. 10:297 Page 22 of 35 http://www. Quantitative considerations Consider two groups of patients who represent two extremes Without radical treatment group A has an 80% relapse rate (eg some soldiers who fought in the Pacific in the Second World War) Without radical treatment group B has a 20% relapse rate (eg some soldiers who fought in the Korean War) Assuming a fixed fractional proportion of relapses and no acquisition of immunity. highly effective.133]. This hypothetical example simply points out that the apparent differences in primaquine “resistance” may reflect differences in the biology of the parasite rather than drug susceptibility per se.5 mg base/day) were applied initially only in Oceania. and all tropical strains susceptibility. a greater proportional reduction in activatable hypno- This highlights the need for better discrimination of the zoites to prevent all relapses.malariajournal. Meanwhile most countries adopted the 15 P.com/content/10/1/297 22. These soldiers were followed for eight showed that concurrent quinine and pamaquine (plas.25 mg base/kg (15 mg adult dose) reduces the number of viable activatable hypnozoites (VAH) by 90%.86] and and July 1925 Sinton compared two different quinine studies during and after the Second World War [47] regimens in the treatment of falciparum malaria in Brit- provided further support for this notion.but it is not Asia). It remains possible that all temperate “strains” are at all clear if there has been any significant change in equally sensitive to primaquine. and the phar- macokinetic-pharmacodynamic basis of radical Vivax malaria following falciparum malaria treatment. One particular area of therapeutic relevance In July.

com/content/10/1/297 detailed below point to these vivax episodes being primary attack.cynomolgi (the primate malaria “equiva- has been that “sporozoite populations of all strains of lent” of P. There are relatively few recent The temperate zone P. independent of season). Most informative are stu- Further south in temperate climes P. previously it is difficult to understand how multiple zoites. vivax.e. 10:297 Page 23 of 35 http://www. ous peoples living in malaria endemic areas and the and most are now agreed that reinfection from the blood latency periods observed in malaria therapy patients and stage back to the liver to produce secondary tissue stages returned soldiers (i. This inoculated. Shortt and Garnham initially suggested that this sing of a mosquito protein [143]. This remarkable efficiency suggests that muli [141. stage. clock mechanism. Subsets of these dormant pre-erythrocytic stages are preprogrammed to resume development at different times The periodicity of relapse and. very long intervals between relapses (four relapses Under this conjecture then perhaps failure to relapse early occurred with preceding latencies exceeding six months.142]. evoke the sequence of Various theories to explain the remarkable periodicity of relapses that characterize sporozoite-induced infections Plasmodium vivax infections have been proposed [140] with the aforementioned plasmodia” [82]. Much of the theorizing preceded discovery in proves that long-latency does occur with the tropical fre- 1982 of the dormant or persistent liver stage-now called quent relapse phenotype. and relapsing simian malarias such as series [82] he noted that “The mean days separating P. bite was five (one to nine) and 11 of the 39 relapses in this latency “zoites” then long latencies would only be evident group (28%) occurred more than six months after the if the sporozoite inocula were very small (otherwise there initial infection.White Malaria Journal 2011. longitudinal observations following a single infected mos- fortably with a simple preprogrammed biological clock quito bite (Figure 8). The number of relapses varied con- conjecture in which each sporozoite has a programmed siderably. and its emergence and subsequent treatment vals gradually lengthened. relapses could occur at regular intervals with generally lated sporozoites (tachyzoites. In fact they were similar to unnaturally large numbers of sporozoites. by Schmidt) infected with P. Coatney realized the intervals from primary infection to early relapse which this and so. In the seven volunteers who were not reinfected latency. As discussed including reinfection of liver cells from released mero. undergo partial development to the resting hypnozoite ciparum inoculation. some activation or feedback mechanism must operate in sues from blood stage parasites. fal. include a subpopulation that completes each of the first four attacks (primary and first three development promptly and is responsible for the early relapses) were essentially identical in infections treated . A recent suggestion is that mosquito bites them- discovery of the pre-erythrocytic developmental stage in selves might provide the trigger . the Rhesus monkey accepted theory (as described. The interval from one relapse to the next would almost invariably be one or more tachyzoites in the was remarkably similar but overall the inter-relapse inter- inoculum. artemisinin derivatives have been used as these drugs are lation but the first relapse occurred 8-9 months later. In bird malarias there is reinfection of tis. ovale. This observation sits uncom. eliminated rapidly and do not suppress or delay the emer- Although the interval ("latency”) between the primary gence of subsequent relapse [107. and disputed. This is difficult to might also occur in the primate malarias [59-61]. The generally the primate model (Figure 9). intrinsic differences in latency periods of the inocu. These patterns of relapse are also illustrated well in zoite the biological clock model was refined.145]. cynomolgi. Much of the infection and first relapse for this “Madagascar phenotype” early volunteer data with tropical “strains” was confounded was long (8-10 months). P. but the available evidence confirms the acquired in late summer or autumn could coincide with remarkable periodicity documented in the volunteer stu- vector emergence in the following late spring or summer. vivax usually had an incubation data on relapse patterns in frequent relapse “tropical” period of 8-9 months so emergence of an infection vivax malaria. the hypnozoite [65-69]. in his classic series of 204 sporozoite-induced occurred in some Madagascar/St Elizabeth phenotypes infections with the Chesson strain [75]. the subsequent inter-relapse partly by slowly eliminated drug effects and inoculations of intervals were short (Figure 7). away from seasonal mosquitoes and does not take place in the primate malarias.144. he made detailed and all Chesson phenotypes. Importantly there could then be would obscure any later emergence of bradyzoites) [140]. bradyzoites). dies with the “Chesson strain”.malariajournal. However reconcile with the similarity of latency periods in indigen- there is no convincing evidence to support this theory. via this built-in time clock. vivax) [69]. In Schmidt’s detailed longitudinal P.perhaps by parasite sen- the liver. After the discovery of the hypno. simply implies that long-latency (bradyzoites) only were The maximum documented interval was 397 days). and a group of subpopulations that relapses of latent hypnozoites acquired before the P. and activation small inocula (median ~ five hypnozoites) and a simple of dormant parasites by external stresses or seasonal sti. Lysenko et al pointed out that if the inoculated the median (range) number of relapses following a single sporozoites were indeed a mixture of short and long. vivax infections had dies in which anti-malarials such as quinine or the a primary illness two to three weeks after mosquito inocu. Following their seminal addition.

25] (Figure 2). being dis. The proportion is cur- The biology of relapse rently 30% in Thailand [136-138] and 50% in Any theory seeking to explain the remarkable biology of Myanmar [149]. observed in soldiers following intense exposure and 1.malariajournal. Multiple relapses are common. 6. vivax maintains a high degree of genetic diversity tions are followed by a subsequent relapse within 28 days if a rapidly eliminated anti-malarial drug (arte. falciparum malaria illness and the subsequent P. Plasmodium vivax relapse must accommodate the fol.White Malaria Journal 2011. That is that in endemic areas a thought to be relatively small (median 6-10 sporo. a relapse. vivax malaria and the subsequent P. falciparum and P. with Plasmodium falciparum are followed by an epi- sode of P. It is not uncommon in tropical areas for vivax hypnozoites which can be activated by a suffi- children to have four to six relapses at 4-6 week cient stimulus. and volunteer studies all suggest this local strains in neurosyphilis patients (despite larger proportion is a function of sporozoite inoculum. vivax primary infections are followed by mic areas. The relapses in clinical studies conducted in ende- only 5 sporozoites in three monkeys this argues for acti. “the intervals between may also occur after multiple relapses in the tropical relapses were related to size of inoculum. vivax infection. but. tinctly shorter in monkeys inoculated with 5 × 106 spor. vivax infections (up 20% and 80%. 55% in Indian isolates. particularly in and this was undoubtedly a contributor. varying from 18. In Thailand approximately 50% of infec.1 days in infections larger inocu. Mixed species infections in Rhesus monkeys receiving very large sporozoite Mixed blood stage infections with P. This long-latency lums”. 5. despite often low seasonal transmission. Taken together with tropical “strains”. They ascribed this to immunity. to 50%) than in mosquito-borne infections with the apy experience. high proportion of the population have latent P. inocula in the latter). the malaria ther. The epidemiological characteristics suggest that 2. inocula. frequent relapse phenotype [75] (Figure 8). the absence of any relapses following an inoculum of 7.8 days from onset of period of 8-9 months either before the first sympto- patency in infections induced with the smaller inoculum matic infection. or between the first symptomatic and from 18. mic areas are commonly with a genotype which is vation of a proportion of hypnozoites per relapse. Conversely long latencies ozoites up to 5 × 106 sporozoites. hypnozoites (ALH) tion and primaquine is not given [107].com/content/10/1/297 with chloroquine in combination with potentially cura. In these monkey experiments where different interval appears to be normally distributed (mode 28 sporozoite inocula were evaluated.8 to 21. lowing vivax malaria are similar to those between acute P. zoites). 4. and is different to that identified in the primary infection consistent with the earlier observations in soldiers and (48% in Columbian isolates. but another young children. followed by a long interval. Not all P. Even larger numbers of relapses were latent hypnozoites” (ALH) hypothesis. intervals and sometimes more following an incident Four lines of evidence support this “Activation of infection. vivax are underestimated by microscopy. if there are further relapses after the long latent ozoites than in those challenged with 5 × 10 2 period then they occur frequently with short inter- sporozoites. vivax relapse.4 to 22. Relapses of vivax malaria arise from activation of latent sunate) is given for treatment of the primary infec. 16). vals which are very similar to those observed in the pying an intermediary position”. it was noted that. even with cing a relapse after each illness episode in a particu. with recipients of 5 × 104 sporozoites occu. Early relapses reach patency around three weeks these are all relapses (Figure 20). even though sporozoite inocula are explanation is possible. Relapses show remarkable periodicity. Importantly the fraction of people experien. this proportion is insufficient lar location appears constant to explain the 30% to 50% of patients in SE Asia who . sensitive PCR techniques. The intervals between the acute P. A remarkably high proportion of acute infections sporozoites were not studied in the primate model. It is unfortunate that inocula between 5 and 500 8. Elsewhere Swellengrebel and de Buck [17] noted that relapse rates the probability of relapse generally varies between were higher in naturally acquired P. Animal experiments. weeks for the Madagascar strain [24. It is also interesting and perhaps relevant that in ende- 3. infection and the first relapse. In long-latency phenotypes there is commonly a tive agents. 1. malaria therapy patients of a fixed fraction of relapses 61% in Thai and Burmese isolates. after starting treatment which suggests emergence from the liver at least one week earlier. 10:297 Page 24 of 35 http://www. P. although there was no clear difference in the number of Sometimes there are several short interval relapses relapses between monkeys inoculated with 5 × 102 spor. and 71% in East following each illness episode in vivax malaria (Figure Timor isolates) [146-148].

The most plausible explanation for these find- tively uncommon (overall in Asia 6. 10:297 Page 25 of 35 http://www. . ciated with a separate P. In the published literature not one of the 45 patient at the time of acquiring P. The figures in brackets are the number of patients studied [136-138]. vivax being infection and the subsequent P. falciparum ings is that the majority of these P. The interval between the primary P. vivax malaria strongly more rapid). soldiers with brief periods of exposure in SE Asia.com/content/10/1/297 ŝŚLJĚƌŽĂƌƚĞŵŝƐŝŶŝŶͲWŝƉĞƌĂƋƵŝŶĞ ;ϴϲͿ ƌƚĞƐƵŶĂƚĞͲDĞĨůŽƋƵŝŶĞ ;ϭϲϲͿ DĞĨůŽƋƵŝŶĞ ;ϵϮͿΎ ŚůŽƌŽƋƵŝŶĞ ;ϭϬϬͿΎ ƌƚĞƐƵŶĂƚĞͲŵŽĚŝĂƋƵŝŶĞ ;ϵϭͿ ƌƚĞŵĞƚŚĞƌͲ>ƵŵĞĨĂŶƚƌŝŶĞ ;ϭϲϬͿ YƵŝŶŝŶĞ;ϱϴͿΎ ƌƚĞƐƵŶĂƚĞ ;ϮϭϲͿΎ YƵŝŶŝŶĞ;ϭϭͿΎ .6% of P. vivax hypnozoites are activated by acute falci- patients or healthy subjects with gametocytes of both parum malaria. Persuasive support.malariajournal. experience vivax malaria following falciparum malaria species in the blood at the same time.ĂůŽĨĂŶƚƌŝŶĞ ;ϭϬͿΎ Ϭ ϭ Ϯ ϯ ϰ ϱ ϲ ϳ ϴ ϵ tĞĞŬƐ /ŶŝƚŝĂůƚƌĞĂƚŵĞŶƚ Figure 20 The median (IQR.White Malaria Journal 2011. falciparum and P. may occur in low transmission areas it is implausible that ing evidence that these are P. In fact finding vectors groups have much lower rates of P. vivax inoculation within one or ogy studies in which single anopheline mosquitoes have two days. This is also supported by the rarity of finding latent P. age less than one infectious bite per year. range) Intervals between acute P. These cies should also carry the other. If these mixed spe. vectors trapped in Thailand contained both malaria spe. falciparum inoculations would be asso- simultaneously acquired infections comes from entomol. fal- with both P. vivax episodes arise sporozoite positive mosquitoes (17 of 258) also contained from hypnozoites which were latent in the liver of the P. vivax sporozoites is rela. vivax following P. individually examined malaria positive wild anopheline The remarkable similarity of both the timing of the P. There is other cies infections resulted from simultaneous inoculation supporting anecdotal evidence from travellers and from then 30 to 50% of anopheline vectors carrying one spe. vivax malaria (in red) and the subsequent vivax malaria episode in patients studied in Thailand following different anti-malarial treatments. vivax relapses and not over 20% of P. particularly when individuals receive on aver- been examined for both species [150]. falciparum (ALH). Development rates [138]. Although space-time clustering of infections suggests this is a relapse (Figure 20). vivax recurrences and their variance strongly suggest that cies [150]. ciparum. falciparum in the mosquito are also slightly different (P. falciparum malaria (in green) and acute P. * reinfection excluded. vivax).

If the “cross” took place sev. vivax episodes were 3. vivax relapses [146-148]. Relapse rates were particularly high in If P.154]. This would explain satisfacto. vivax malaria intense exposure. However in the causes vivax malaria relapses then it seems that a signifi- majority of relapses the parasites are clearly genetically cant systemic illness is necessary for this activation. it was widely relapse is homologous or highly related with the primary believed that a variety of external stresses could bring infection. It was even taught in close “races” between different genotypes to reach some textbooks of tropical medicine that Cinchona alka- patency commonly result in gametocyte genotype mix. In these cases 4. eral cycles of infection previously then subsequent infec- tions may contain highly related parasites through Mechanisms of hypnozoite activation successive interbreeding between related siblings. By analogy. In unrelated. and induced hypoxia.White Malaria Journal 2011. kine responses) or malaria specifically could provide the ficial infections which follow inoculations with 5-10 activating or inhibitory necessary stimuli to generate times more sporozoites should have higher. In approximately one third of P. Nevertheless it is der of Thailand. or could result from latency phenotypes particularly if the interval is eight recombination in the anopheline vector with the pro. relapses in the volunteer studies). vivax hypno.malariajournal. simulated altitude.all exceeding six months after their preceding latent hypnozoite(s). bird haemosporidian parasite Leukocytozoon are pro- zoites’ progeny won the race to patency against the het. vivax malaria was acquired. falciparum malaria activates latent P. periods. 10:297 Page 26 of 35 http://www. The incidence and number of relapses Periodicity can be generated in several ways in biologi- depends on the number of sporozoites inoculated. Illness generally (and the associated cyto- relapses derived from the inoculated infection then arti. It is not uncommon to encounter patients vivax relapses studied in South East Asia. quent relapse phenotypes can remain dormant for long simultaneous inoculation). vivax in North Africa or Italy) were appar- patients studied in this area. The relapses of vivax malaria in the interesting that soldiers in field hospitals in the Mediter- babies’ mothers were usually genetically different to ranean region between 1940 and 1945 (who had those which caused the primary infection. In the one third of patients in whom the the first half of the twentieth century. soldiers who were immunologically naïve and underwent zoites then there is no reason why P. Natural versus artifical infection relapse rates associated with malaria. -10 months).156]. Heterologous genotypes rates of relapse. two thirds of P. Long-latency also occurs in the tropical frequent relapse where the original genotype was not detected in the “Chesson” P. or the homologous infection’s hypno. vivax infection of life in their babies malaria (presumably a relapse) if they had pneumonia or were usually of the same genotypes as those which hepatitis compared with trench foot [155. However formal studies to provoke mosquito). This proves that some hypnozoites of fre- duction of genetically related sibling sporozoites (i. In our caused the initial infection [151]. the SPf66 malaria vaccine [157]P. If all relapses derived from the most should not do the same. Overall it seems most . or its hypnozoite(s) were activated but quito bite in Coatney’s series had relapses of the Ches- their progeny were outcompeted by the earlier activation son strain of P. not yield convincing results [153. but not with minor illnesses (S Higher rates of vivax relapse in indigenous compared Lee. Obviously the infants own series of children seen every day for over 18 could not have any previously acquired latent hypno. whereas the relapses which ently considerably more likely to experience vivax followed the first P. loids should be given before surgery to pre-empt a tures in relapses (which may then recombine in the relapse of malaria. the malaria returning from malaria endemic areas where tropical parasites isolated are either identical or closely geneti. which included forced route comes from observations in mothers and their infants marches. vivax with variable but long latency or more rapid growth of the progeny of the activated periods .com/content/10/1/297 2. ALH hypothesis. as in other acquired P. did living in a malaria endemic area on the north-west bor. not lower periodic relapses in vivax malaria. More If acute malaria activates latent hypnozoites and thereby work on this important area is needed. relapse [75]. so it seems that a sub- with artificial infection would also be explained by the stantial systemic stimulus is required. personal communication). recent inoculum then there should be no relationship rily the finding of heterologous genotypes in one half to between intensity of exposure and number of relapses. Further support for the ALH hypothesis relapses of vivax malaria. either there were no latent hypnozoites (as in on a relapse of malaria [140]. It is evident then that of long migratory flights [152]. phenotypes are prevalent with relapses more than 3 cally related to the primary infection. If all cal systems. voked by the stresses of egg laying and the exhaustion erologous hypnozoites’ progeny. vivax phenotypes malaria recurrence then either the inoculated infection Four of seven volunteers receiving a single infected mos- did not relapse. Relatedness could months after either a primary infection or return to the occur if there is little genetic diversity in the area where non-endemic area (of course these might also be long- P. months on the Thai-Burmese border during a study of zoites in their livers to be activated by the illness.e.

If there are some hypnozoites which fail to be activated these may be activated at a later date by a subsequent malaria infection. Either results is very complex and difficult to reconcile with the effi. A a negative feedback mechanism. Half the newly acquired infection sporozoites (blue) develop into preerythrocytic schizonts and half become dormant as hypnozoites (the estimated proportions for tropical “strains”) [59. The different genotypes are denoted by different colours (red and white).malariajournal. small numbers of hypnozoites (i.e. The ensuing illness activates some of the remaining hypnozoites (the same fraction as were activated initially) and relapses continue until either the number of hypnozoites is exhausted or some fail to be activated. Importantly the proportion of Ϯ͘WƌŝŵĂƌLJŝŶĨĞĐƚŝŽŶ ϯ͘WƌŝŵĂƌLJŝůůŶĞƐƐ ϭ͘/ŶŽĐƵůĂƚŝŽŶ ϲ͘&ƵƌƚŚĞƌĂĐƚŝǀĂƚŝŽŶ ϱ͘ZĞůĂƉƐĞ ϰ͘. at the time of infection (sporozoite inoculation) the individual already has hypnozoites of two different genotypes acquired from two previous inoculations which are latent in the liver. in several relapses at regular intervals. or propose simply a bution of latencies alone is inadequate to explain the relatively broad temporal distribution of latencies (ana- observed phenomena. 10:297 Page 27 of 35 http://www.White Malaria Journal 2011. a logous to seeds germinating or eggs hatching).ŽŵŽůŽŐŽƵƐŐĞŶŽƚLJƉĞ ϭϬϮ Ϭ Ϭ Ϯ ϰ ϲ ϴ ĂLJƐ Figure 21 Proposed mechanism and sequence of Plasmodium vivax relapse activation in a malaria endemic area. By chance the progeny of one of the preexisting latent hypnozoites reach pyrogenic densities before the progeny of the recently inoculated hypnozoite (inset shows the logarithmic growth of the three genotypically different infections-vertical axis shows number of parasites in the body). each activated by cient use of relatively small sporozoite inocula (median the preceding illness. The consequent febrile illness then suppresses further multiplication of the blood stage infection so that the progeny of the other two prerythrocytic schizonts may not reach transmissible densities. so that they do not and that each symptomatic episode provides an activa.com/content/10/1/297 likely that the illness associated with the infection ~10 sporozoites). In an illustrated example. vivax relapses (Figure 21). In this example there is one hypnozoite of each genotype and each is activated by the illness and each develops into a pre-erythrocytic schizonts. which may occur with simple clock mechanism with a single unimodal distri. vivax fever pattern in relation to asexual parasite development). Illness associated with the blood stage infection activates a small fraction of the hypnozoites (inset shows a “classic” P. as explained previously. . illness being the negative feedback.ĞƚĞƌŽůŽŐŽƵƐŐĞŶŽƚLJƉĞ ϭϬϭϬ Ͳϭ WLJƌŽŐĞŶŝĐĚĞŶƐŝƚLJ;ϭϬϬȝ> Ϳ ϭϬϴ ϭϬϲ ϭϬϰ .LJƉŶŽnjŽŝƚĞĂĐƚŝǀĂƚŝŽŶ ŽĚLJƉĂƌĂƐŝƚĞďƵƌĚĞŶ ϭϬϭϮ . For efficient yet periodic activation of itself is the activator in P. all activate together) it is necessary either to hypothesise tion stimulus which may give rise to the next relapse. and a multiplicity of different latencies with a single harmonic low individual susceptibility to activation.69]. Equally.

If the duration of pre-erythrocytic develop- vivax malaria following P. but the illness then is indeed a biological clock (which is most evident in activates further hypnozoites.19. Garnham observed a ten-fold reduction in the number of nozoite activation declines with increasing disease con. This would explain the high rate pared with the tropical strains may reflect the high natural of vivax malaria (presumably relapses) following falci. 10:297 Page 28 of 35 http://www. This can then be subdivided into the pro- infection follows activation stimulus such as a malaria portion of sporozoites which activate immediately and illness). vivax. In this from activation and how much was from cell death [68]. One possi- the subsequent relapse. hypnozoites in serial liver biopsies over a nine month per- trolling immunity. but it is not clear how much of this reduction resulted probability that the relapse is asymptomatic. ble mechanism contributing to regular short interval zoites (half way through their sleep) were absolutely (three weeks) relapses is a malaria illness related tem- refractory at this time.27-29. no long-latency P. Recent studies in murine malaria indi- latent and not immediately activatable. It is interesting to specu- latent until either they die (for example when the host late that the large proportion of sporozoites dedicated to hepatocyte dies) or they are activated by a systemic ill. It is likely that once hypnozoites porary halt to liver-stage development (mediated by do become activatable. If this is correct it vivax. or not activation does occur. and the relatively low number of relapses com- ness such as malaria.e. tions of patients who experience multiple relapses (Fig. sporozoite. It follows that the number of immediate the hypnozoites become activatable. but that there is a initial infection.White Malaria Journal 2011. which fits with the observed fixed propor. two populations of hypnozoites. activation among the remaining hypnozoites. P. 8-10 months after the primary mines the length of the interval before the hypnozoite infection) should usually be of a similar genotype to the becomes susceptible to activation.com/content/10/1/297 susceptible hypnozoites activated would be fractional. first sporozoites. as seems likely. heterologous. there are clearly at least genetic basis for this regulation may be difficult to find. temperate strains) determining latency in Plasmodium quent short inter-relapse intervals. and immunity increases the iod. . respect it is interesting that in Thailand the incidence of The hypnozoite can be considered as an unactivated symptomatic P. vivax peaks in childhood [106]. If there are subsequent relapses i.vivax transmission.e.160]. an important regulatory role in controlling hypnozoite tomatic malaria but critically did not affect the timing of activation and pre-erythrocytic development. as the stimulus to hyp. the subsequent temporal distribution of susceptibility to For temperate strains of P. clock.79. long-latency P. which is then lifted with clini- tively low rate of spontaneous activation in order to cal recovery. The study of cate a central role for iron sequestration in controlling Cooper et al. If the ALH hypothesis is correct then the that with small inocula it is quite possible for all sporo. that there is a background rela. falciparum may contribute hypnozoites. The trigger could be either which follow the first relapse after the latent period (i. is informative in this respect [159. wastage of hypnozoites in hepatocytes which die before parum malaria. vivax phenotypes lies in the temporal relapses) or for all to result in hypnozoites. a positive activation stimulus or removal of inhibition. in which blood induced homologous (St pre-erythrocytic development through malaria illness Elizabeth strain) infections were induced 120 days after induction of the iron regulatory hormone hepcidin infectious mosquito bites. The eral investigators previously. Plasmodium vivax in temperate zones has clearly ure 16). latency. whereas relapses at other times could separate sensing mechanism which determines whether be heterologous. This clock (which could be an intrinsic parasite follows that first relapses which occur with a long. this puts activation at the significantly to P. The blood inoculations reliably gave rise to symp. at with a short periodicity) then these could be genetically least initially.158]. This suggests that the hypno. one becoming activata. as suggested by sev. falciparum malaria shows a ment of the liver stage is similar for sporozoites and weaker age relationship. In experimental P. It is possible that iron availability also plays [35]. particularly in time or shortly after presentation with acute malaria (of young adults. vivax the first relapse after the long In summary this ALH hypothesis proposes that there latent period is usually spontaneous. Thus for the in the ex-vivo hepatocyte culture systems [70]. key biological difference between frequent relapse and zoites to develop immediately (early infection. vivax) and another remaining tive differences. as it could well reflect subtle quantitative rather qualita- ble early (as for tropical P. relapses per mosquito inoculation will decrease with cynomolgi bastianelli infection in the Rhesus monkey increasing age in endemic areas. or could reflect a host-parasite interaction) deter- latency interval (i. but P.e. reduced iron availability). It also follows any species). through this mechanism. Some of these hypotheses may be testable account for the distribution of latencies.malariajournal. accounting for the subse. It also may leave a significant proportion of evolved to adapt to the long winters across the Northern unactivated (but activatable) hypnozoites which remain hemisphere [17. and all the distribution of susceptibility to activation among the hypnozoites to become latent (no early infection.

and the use of pri- with longer transmission seasons this may be difficult to maquine in “combination therapies”. and immune responses are amongst the frequent relapses. These cases provide an important epidemio- hypnozoite has become susceptible. this may explain the discern [19]. In endemic areas of South-East Asia.com/content/10/1/297 This mechanism may activate spontaneously once the [108-112]. some of which may com- Implications for epidemiological assessment prise very large numbers of parasites (> 1012) [162]. this figure resistance is approximately one quarter to one third. If the de-novo resistance is high grade then the resis- early relapse. falciparum infections. if patients who acquire falciparum Implications for the emergence of anti-malarial drug malaria are representative of the population. Of course the resis- and his colleagues [75]. This is because a primary illness erologous or drug sensitive homologous (sibling) parasites. Together with the lower biomass. that long-latency phenotypes are present in many tropi- dual probability of activation for each hypnozoite is low.malariajournal. The factors determining de-novo selection of anti-malarial city of P. essential prerequisite for therapeutic assessments. 10:297 Page 29 of 35 http://www. expanding intra-host population of de-novo resistant para- line vectors was an obvious clue. In temperate areas. parasite biomass. the early spring-summer wave of quent recrudescence. vivax frequent relapse patterns may still be tion is effectively in a race with the relapse-either of het- observed (Figure 22). fre- eling will provide valuable insights into which activa. The net result would be indistinguishable not be transmitted onward in infections in which the from the epidemiological pattern of frequent relapse relapse becomes patent before the recrudescence would vivax malaria (Figure 22). Despite a resurgence of is much more likely with an external systemic illness interest in malaria. If both types are prevalent in tant parasites are less drug-sensitive so they will have a the same area then identifying phenotypes from illness “second chance” to recrudesce later if the same drug is patterns becomes even more difficult. The presence of long-latency phenotypes slower emergence of low-grade anti-malarial drug resis- may only be evident in travellers and soldiers who tance in P. and spontaneous logical signal. and treat- demonstrated clearly in the studies of Robert Coatney ment usually follows soon afterwards. In P. parasites (pattern of drug exposure. but once susceptible activation the P. The relapse therefore pre-empts the vivax malaria which preceded the appearance of anophe. falciparum. The much lower parasite biomass therapy. as is more intervals even though all the parasites are of the long. with long-latency P. this large knowledge the infected hepatocyte (which is very sensitive to sys. and why in areas with long. This implies that trol and elimination planning. Mathematical mod. do not receive Slowly eliminated drugs interefere with the resistance primaquine. quency and stability of the genetic or epigenetic mechan- tion-inhibition model best fits the observed malaria ism. [161]. then resistance can- latency type. but in tropical regions sites. and then return to a non-endemic area and protection provided by relapse. and the evaluation of people living in vivax endemic areas commonly harbour novel interventions such as vaccines. It is quite possible temic inflammatory responses). and the belated recognition of the such as malaria. in P. and it may be used for treatment of relapse. This is because parasite multiplication with the tropical frequent relapse phenotypes was falls rapidly once symptoms have developed. In If the ALH theory is correct it also explains why relapse malaria. Activation must involve signaling via importance of Plasmodium vivax. usual in anti-malarial drug resistance. recrudescence is necessary for onward transmis- phenotypes in tropical malaria endemic areas may be sion of a de-novo resistant mutant parasites’ progeny difficult to characterize. vivax of the “Madagascar” or “St Resistant parasites will only be transmitted if their progeny Elizabeth” phenotype may activate previously acquired reach transmissible parasite densities before the relapse latent hypnozoites (of similar phenotype) -giving an does. gap seems to have gone unnoticed. volunteer. and epidemiological information. The illness caused by the relapse could tant parasites may expand in numbers rapidly and may not then activate further latent homologous or heterologous be impeded by the relapse. cal areas (Figure 17). vivax compared with P. vivax relapses is derived from the sequential drug resistance are likely to be similar across all malaria activation of hypnozoites by illness. Defining these patterns is an allowing accumulation of latent but “activatable” hypno. However if the first step in hypnozoites. There is uncertainty over the true epide- activation presumably usually explains the first relapse miology of relapse patterns over a large proportion of after a long latent period. Importantly the indivi. vivax malaria the de-novo recrudescent infec- latency P. vivax endemic world. latent but “activatable” hypnozoites. but treatment may change. The periodi. con- zoites after each sporozoite inoculum. spend a brief period in the endemic area. vivax malaria makes de-novo selection less likely than in P. such strengthening -all of which reduce the chance of subse- as Southern Europe. fitness cost) [161].White Malaria Journal 2011. Chloroquine has a slow relapse many months later without re-exposure multiphasic elimination phase and suppresses the . impossible to discern the long-latency phenotypes primaquine may be added. Thus several relapses may follow at short resistance is a small reduction in susceptibility. The converse -long-latency have (Figure 23).

In summary seasonal transmission. early relapse provides a brake on the emergence and spread of low-grade resistance in Plasmodium vivax by Practical implications pre-empting recrudescence. In the lower panel the long-latency phenotypes are present. Three relapses occur with similar periodicities. If the ALH theory is correct then the epidemiology of malaria relapse and the biology of interaction between Implications for genetic diversity the species need reconsideration. These are sequentially activated as proposed in Figure 21. and a later randomly activated relapse occurs 8 months later. This illustrates the difficulty in excluding the presence of long-latency phenotypes in malaria endemic areas. vivax phenotypes. will ingest gametocytes of different genotypes. tive method of increasing the likelihood that a mosquito ent profile of elimination. The patient has hypnozoites from three preceding inoculations present at the time of illness from the newly acquired (red) infection. vivax parasites. 10:297 Page 30 of 35 http://www. This must be an important relapse providing greater suppression of the sensitive contributor to the relatively high degree of genetic compared with the resistant parasites and they therefore diversity in P. vivax often found in areas with very low reduce the delay on resistance emergence.White Malaria Journal 2011. and provides longer suppres. thereby sion of vivax relapse.malariajournal. The different colours and symbols represent different genotypes in two hypothetical infections. Activation occurs as above and the long-latency relapse emerges nine months later. The residual drug levels reduce facilitating meiotic recombination between genetically potential interference with resistance emergence by unrelated P. relapse of sensitive parasites for approximately two with asexual stage development this provides an effec- weeks (from two to six weeks).com/content/10/1/297 NewinfectionRelapses Tropicalfrequentrelapsephenotype Longlatencyrelapsephenotype NewinfectionRelapses 0123456789 Previously acquired Months hypnozoites Figure 22 Potential similarity of relapse patterns with the long-latency and frequent relapse P. Mefloquine has a differ. vivax malaria in the Indian sub-continent and further types reaching patent parasitaemia at similar times. The initial relapse patterns associated with the two different phenotypes are identical. vivax occurs simultaneously cines may also need reconsideration (Table 3). . The upper panel shows the relapse pattern where the frequent relapse phenotypes are prevalent. As west then current methods of assessing drugs and vac- gametocytogenesis in P. If long-latency Plasmo- Activation of hypnozoites from different preceding dium vivax still contributes a significant proportion of inoculations will commonly result in two or more geno.

Genotyping should assist in assessing long-latency relapse. A substantial proportion of the population in P. Reducing P. It is very difficult to exclude the presence of long-latency P. Spontaneous or activated relapse followed by asymptomatic parasitaemia may be an important source of P. and we do not 100. Taking a quantitative any treatment effect) suppresses multiplication. A single radical treatment for all malaria infections may be justified in areas where both parasites are prevalent (i.vivax infections and reduce P. malaria control interventions which are effective will not The apparent radical curative activity of primaquine prevent relapses emerging for months or years. organism load and immunity. ACT + radical primaquine regimen) [143]. vivax phenotypes in studies conducted in vivax endemic areas. Some of these may derive from inoculations which were not followed by any illness. 2.malariajournal. multiplies while the sibling drug sensitive parasite population declines (green) [160].com/content/10/1/297 activation. Long-latency phenotypes may be prevalent over a much wider area of the tropics than currently thought. The same Figure 23 Relapse pre-empts the emergence of de-novo anti. The proportion of acute 104 falciparum malaria infections. This would result in relatively low relapse rates. approach. Lower dose regimens may have useful efficacy in will result in less illness and therefore less hypnozoite this context. 7. Assessment of the efficacy of interventions requires characterization of the prevalent relapse phenotypes. De-novo drug resistance is a rare event stage infection appear to affect the responses to hypno- and usually there is only one mutant resistant parasite which zoitocidal treatment i. 4. although would be expected to improve as malaria transmission their number will reduce as the reduced transmission falls. 6. then relapse becomes the predominant cause of vivax malaria. It is possible that much of the variance in responses to 1 primaquine is explained by differences in rates and bur- 0 1 2 3 4 dens of latent hypnozoite carriage and degree of immu- Weeks nity and not variation in drug susceptibility. factors which affect therapeutic responses in the blood malarial drug resistance.e. If the majority of relapses in endemic areas not be comparable to studies in travelers. Slowly eliminated approach to assessing 8-aminoquinoline radical curative anti-malarial drugs reduce this protective effect by reducing multiplication of the relapse parasites more than multiplication of activity based on hypnozoite burdens may be a valuable the de-novo resistant parasites. Studies in derive from heterologous latent hypnozoites then adults may not be comparable to studies in children. Long term follow up (minimum one year) Table 3 Epidemiological implications 1. The proportion of genotypically different (heterologous) relapses will fall as transmission intensity falls . 3.e. vivax transmission. If relapse rates exceed 50%. which are followed by P. 10:297 Page 31 of 35 http://www. 9. organism phe- sensitive parasites) becomes patent and the consequent illness (and notype.White Malaria Journal 2011. vivax may be a better indicator of the prevalence of 102 latent hypnozoite carriage.vivax transmission. Reducing P.000. 5. Only a highly resistant parasites’ progeny (red We have tended to consider 8-aminoquinoline efficacy line) can reach transmissible densities in blood (total numbers circa only from the perspective of the drug. vivax endemic areas harbours latent but activatable hypnozoites. However any effect on the incidence of clinical disease would probably be delayed because reducing falciparum malaria will also reveal vivax malaria by lifting suppression in mixed infections.000 in the body) before the relapse (comprising drug take into account either organism load. and a reduction in vivax incidence will reduce immunity. Assessments of radical curative activity where long-latency phenotypes are prevalent require one year’s follow-up. vivax transmission will have a smaller than currently predicted effect on the incidence and prevalence of vivax malaria initially. Patients with very large liver burdens of hyp- nozoites from either a very heavy inoculation or multi- ple inoculations and little or no immunity (such as Radical treatment therapeutics soldiers) would be expected to have a larger number of In therapeutic assessments a follow-up period of six relapses than travellers who have a brief period of expo- months or less may miss a significant proportion of the sure. Mass drug administration with radical cura- Total parasites Relapse tive regimens (currently primaquine is the only option) 1010 would be the only way to eliminate this reservoir of infection quickly. Studies of radical curative activity in soldiers may relapses.falciparum transmission may reduce the incidence of P. or host immunity. 8. Epidemiological assessments in older 108 children and adults in endemic areas may underestimate the burden of vivax malaria as partial immunity (and 106 premunition) will ameliorate disease severity and may lead to reduced activation of relapses.

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