Nicholas J White

“ Interest in the long-latency phenotype revived during the early 1950s as P. vivax malaria which had a long-latency was an important problem in soldiers fighting in the Korean war [61] . During this period patients who had received very heavy inoculations, typically soldiers from the Second World War, would return to clinics for many years complaining of recurrences of malaria. The long latencies and the long relapse intervals in some infections and the multiple relapses in others despite anti-malarial treatment gave rise to the old saw that "you never got rid of malaria". ”

“ This mechanism may activate spontaneously once the hypnozoite has become susceptible, and spontaneous activation presumably usually explains the first relapse after a long latent period, but once susceptible activation is much more likely with an external systemic illness such as malaria. Activation must involve signaling via the infected hepatocyte (which is very sensitive to systemic inflammatory responses) . Importantly the individual probability of activation for each hypnozoite is low, allowing accumulation of latent but "activatable" hypnozoites after each sporozoite inoculum. ”

“ There is uncertainty over the true epidemiology of relapse patterns over a large proportion of the P. vivax endemic world. Despite a resurgence of interest in malaria, and the belated recognition of the importance of Plasmodium vivax, this large knowledge gap seems to have gone unnoticed. It is quite possible that long-latency phenotypes are present in many tropical areas (Figure 17) . Defining these patterns is an essential prerequisite for therapeutic assessments, control and elimination planning, and the evaluation of novel interventions such as vaccines. ”

“ 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. ”

“ In temperate areas, such as Southern Europe, the early spring-summer wave of vivax malaria which preceded the appearance of anopheline vectors was an obvious clue, but in tropical regions with longer transmission seasons this may be difficult to discern [ [16] . The presence of long-latency phenotypes may only be evident in travellers and soldiers who spend a brief period in the endemic area, do not receive primaquine, and then return to a non-endemic area and relapse many months later without re-exposure [ [91] - [94] . These cases provide an important epidemiological signal. ”

“ Today there is a tendency to regard all P. vivax together as a single homogenous species, but the human malaria therapy and volunteer studies showed that there was substantial phenotypic variation between Plasmodium vivax "strains". There has been corresponding taxonomic debate over how these "sub-species" should be defined (Figure 6) . Studies conducted over fifty years ago indicated that incubation periods, numbers of merozoites per blood schizont, antigenic relationships, intrinsic drug susceptibility, virulence, and relapse intervals all differed between "strains". ”

“ Plasmodium vivax is a sophisticated and resilient malaria parasite which was once prevalent over much of the inhabited world. It has receded from North America, Europe and Russia, but in the tropics vivax malaria remains a major cause of childhood illness. In most endemic areas, P. vivax cohabits with Plasmodium falciparum. Mixed infections with the two species are common. P. vivax is more difficult to control and eliminate than P. falciparum because of its tendency to relapse after resolution of the primary infection. ”

“ It is generally recognized that asymptomatic P. vivax infections are common in endemic areas but their overall contribution to P. vivax transmission in malaria-endemic areas, and the importance of relapse in maintaining these asymptomatic infections has not been characterized adequately. However the malaria therapy experience suggested that asymptomatic infections were a very important source of infection [145] . An effective vaccine which did not confer long-lasting immunity and required frequent boosting might provide a selective pressure towards longer latency. ”

“ Higher rates of vivax relapse in indigenous compared with artificial infection would also be explained by the ALH hypothesis. The incidence and number of relapses depends on the number of sporozoites inoculated. If all relapses derived from the inoculated infection then artificial infections which follow inoculations with 5-10 times more sporozoites should have higher, not lower rates of relapse. Relapse rates were particularly high in soldiers who were immunologically naïve and underwent intense exposure. ”

“ If acute malaria activates latent hypnozoites and thereby causes vivax malaria relapses then it seems that a significant systemic illness is necessary for this activation. In the first half of the twentieth century, it was widely believed that a variety of external stresses could bring on a relapse of malaria [124] . By analogy, relapses in the bird haemosporidian parasite Leukocytozoon are provoked by the stresses of egg laying and the exhaustion of long migratory flights [ [135] . ”

“ The infections were in non-immune adults whereas the burden of vivax disease in endemic areas was in children. Adults in the malaria endemic areas have usually developed significant immunity to a broad range of local parasites which controls symptoms and reduces parasite densities. The artificial infections in the majority of volunteer studies and in malaria therapy followed the bites of 5-10 infected anopheline mosquitoes selected for maximal infectivity based on salivary gland sporozoite loads in sibling mosquitoes. ”

“ Periodicity can be generated in several ways in biological systems. Illness generally (and the associated cytokine responses) or malaria specifically could provide the activating or inhibitory necessary stimuli to generate periodic relapses in vivax malaria. Overall it seems most likely that the illness associated with the infection itself is the activator in P. vivax relapses (Figure 21) , and that each symptomatic episode provides an activation stimulus which may give rise to the next relapse. ”

“ In 1913, Bignami proposed that relapses of malaria all derived from persistence of small numbers of parasites in the blood [22] . Although this theory explained the late recurrences of Plasmodium malariae infections in man, it did not explain several features of P. vivax and P. ovale infections. A much clearer understanding of relapse in P. vivax malaria was to come from Julius Wagner-Jauregg's discovery that malaria could cure neurosyphilis. Between the 1920s and the 1950s thousands of patients confined in mental hospitals with neurosyphilis were treated with malaria (malaria therapy) . ”

“ 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. ”

“ It also suggests that relapses are probably partially suppressed in older patients. Thus both the proportion of infections which relapse and the number of symptomatic relapses per mosquito sporozoite inoculation decline with age. It is likely that immunity and therefore age is a significant confounder in epidemiological assessments based on passive case reporting in many P. vivax endemic areas. In many areas adults are more likely to present to malaria clinics than children. In India the peak age of malaria presentation is often in young adults (and often with a predominance of young males) . ”

“ Studies of radical curative activity in soldiers may not be comparable to studies in travelers. Studies in adults may not be comparable to studies in children. The apparent radical curative activity of primaquine would be expected to improve as malaria transmission falls. Lower dose regimens may have useful efficacy in this context. Long term follow up (minimum one year) of well characterized patients with parasite genotyping in low transmission settings should help to dissect the contributions of pre-existing versus recently inoculated hypnozoites to relapse. ”

“ In East Asia and Oceania a similar pattern pertains today. In East Asia a remarkably high proportion (20-50%) of symptomatic infections with P. falciparum are followed by an infection with P. vivax [121] - [122] . In Ethiopia where early relapse rates in vivax malaria are much lower, this proportion is also lower; 7% [ [123] . The intervals between the onset of treatment for the acute P. falciparum infection and the subsequent P. vivax infection are very similar to the intervals between acute vivax malaria and the first relapse (Figure 20) . ”

“ Several other important observations were made in the artificial infection studies conducted in humans and experimental primates. It was noted that even with a single infected mosquito bite some Chesson strain P. vivax infections relapsed after intervals which were as long as a year after a series of regular "short interval" relapses [60] . These terminal long intervals did not appear to have a fixed periodicity (Figure 8) .
Relapse patterns in volunteers in the USA who were infected by a single mosquito bite with the Chesson strain of P. vivax studied by Coatney et al [60] . ”

“ Taken together with the absence of any relapses following an inoculum of only 5 sporozoites in three monkeys this argues for activation of a proportion of hypnozoites per relapse, and is consistent with the earlier observations in soldiers and malaria therapy patients of a fixed fraction of relapses following each illness episode in vivax malaria (Figure 16) . It is unfortunate that inocula between 5 and 500 sporozoites were not studied in the primate model. ”

“ This implies that people living in vivax endemic areas commonly harbour latent but "activatable" hypnozoites. In endemic areas of South-East Asia, if patients who acquire falciparum malaria are representative of the population, this figure is approximately one quarter to one third. The periodicity of P. vivax relapses is derived from the sequential activation of hypnozoites by illness. Mathematical modeling will provide valuable insights into which activation-inhibition model best fits the observed malaria therapy, volunteer, and epidemiological information. ”

“ This is also supported by the rarity of finding patients or healthy subjects with gametocytes of both species in the blood at the same time. Development rates in the mosquito are also slightly different (P. vivax being more rapid) . Although space-time clustering of infections may occur in low transmission areas it is implausible that over 20% of P. falciparum inoculations would be associated with a separate P. vivax inoculation within one or two days, particularly when individuals receive on average less than one infectious bite per year. ”

“ The effects of age
In malaria endemic areas, such as the north-western border of Thailand, the age profile of P. vivax malaria suggests much more rapid acquisition of immunity than for P. falciparum [101] (Figure 18) . Entomological studies suggested similar transmission rates (at least in terms of measured entomological inoculation rates) so it is likely that relapse contributes to much of this difference. ”

“ A recurrence of malaria (any species) before eight weeks was termed a recrudescence, recurrence from 8-24 weeks was termed relapse, and return of malaria after 24 weeks was called a recurrence. The pattern observed by James in England, Swellengrebel in The Netherlands, and Ciuca in Romania with the Madagascar strain of P. vivax was very similar to patterns of the St Elizabeth and McCoy strains, of indigenous origin, used for malaria therapy in the United States [25] - [28] . ”

“ In natural settings multiple genotypically distinct hypnozoites may be activated but, on many occasions, only one or two genotypes will be detected subsequently at clinical relapse. The other hypnozoites' progeny may reach patency later, or asexual growth may be suppressed by fever, illness, immune response, and treatment such that they never reach patency.
Relapse patterns of P.cynomolgi infections in Rhesus monkeys studied by Schmidt [70] . The infections were induced with different numbers of injected sporozoites as indicated and treated repetitively with chloroquine. ”