Background
Malaria represents a constant burden and public health challenge in approximately 100 countries worldwide [
1].
Plasmodium falciparum is responsible for the largest number of cases and malaria-associated deaths, with extensive research efforts focused on understanding the basic biology and associated pathogenesis of this parasite species.
Plasmodium vivax has also been recognized as a major contributor to the global burden of malaria, and numerous recent reports indicate infections can result in complications with lethal outcomes [
2‐
5]. However by comparison, neglect of
P. vivax is apparent, greatly due to the challenges associated with studying this species, which include the lack of a long-term in vitro culture system or a rodent model that can support the entire life-cycle and re-create critical aspects of
P. vivax infections and disease as observed in humans [
6‐
9].
Plasmodium vivax, as well as
Plasmodium ovale, differ from the three other human malaria-causing species (
P. falciparum,
Plasmodium malariae,
Plasmodium knowlesi) because they can develop dormant liver-stage forms, known as hypnozoites [
10]. Hypnozoites can activate and multiply, causing relapsing blood-stage infections days, months or years after the primary infection. Relapses may result in clinical disease and, importantly, provide the chance for gametocytes to encounter anopheline mosquito vectors and ensure transmission. New insights are needed to treat this liver-stage reservoir to ensure the elimination of relapses and blood-stage infections containing infectious gametocytes. Virtually nothing is known about the biology of relapse infections despite the fact that relapses are thought to be responsible for as high as 96 % of vivax infections in different parts of the world [
11]. It is currently unclear how relapses are similar or different from primary infections, from a parasitological and clinical perspective, and the relative contribution of relapses to clinical malaria has been uncertain.
Understanding the course of primary and relapse infections, and specific mechanisms that result in vivax malaria pathogenesis, disease severity and recovery, are important goals. In particular, the identification of mechanisms that function in the peripheral blood and bone marrow resulting in the onset and recovery of malarial anaemia [
12,
13] and thrombocytopaenia [
14] may aid the search for novel interventions and therapeutic strategies to manage and/or alleviate these common complications. Headway has been made with the study of specimens isolated from human patients living in endemic areas [
15,
16]; however, these studies are mostly restricted to one or a few small blood samples at the time of illness and treatment. Furthermore, the analysis and interpretation of these studies can be affected by uncontrollable variables such as diet, medications, transmission characteristics, co-infections, and other maladies. These factors can confound associations of clinical signs and symptoms. Non-human primate animal models can eliminate a number of these concerns, with experimental plans allowing for the study of specific biological, immunological or pathological processes in a controlled, prospective and manipulable environment.
Non-human primate models have contributed to the broad understanding of malaria biology, pathogenesis and immunity with regards to liver-stage and blood-stage infections, and they have also been instrumental for screening vaccine and drug candidates or specific formulations [
17‐
21].
Plasmodium vivax can be studied directly in New World monkey species, such as squirrel (
Saimiri boliviensis) and owl (
Aotus sp.) monkeys, and these models have been important, for example, in testing vaccine and drug candidates with parasite challenge infections [
22,
23] and, recently, to identify and characterize blood-stage proteomes of
P. vivax infected erythrocytes [
24,
25]. However, due to the small size of these animals (~1 kg), which limits the amount of blood (or bone marrow) available for sampling, and the lack of validated reagents available to evaluate host physiological and immunological responses, they are not ideal for extensive hypothesis testing in relation to malaria pathogenesis, immune responses and recovery processes.
Plasmodium cynomolgi is a simian malaria parasite of Old World macaques that is genetically closely related to
P. vivax [
26,
27] and shares many biological similarities to the human parasite including the preferential invasion of reticulocytes [
28,
29], development of unique infected red blood cell (RBC) structures called caveola-vesicle complexes [
30,
31], and critically, the ability to form hypnozoites that can reactivate and cause relapse infections [
10,
18,
32,
33]. Macaques are closely related to humans, and a large variety of cross-reactive reagents for assessing host responses have been developed since these monkeys are model organisms for infectious diseases [
34]. Young adult macaques (~5 kg or greater) can support longitudinal
Plasmodium infection studies that require repeated sampling within a short time frame.
Macaca mulatta (rhesus monkey) and
M. fascicularis genome sequences [
35,
36] and the genome sequence of several strains of
P. cynomolgi [
26], have been characterized in recent years, enabling basic as well as systems biology studies of host-parasite interactions.
Here, comprehensive analyses of sporozoite-initiated, longitudinal infections of
P. cynomolgi in rhesus macaques are presented with the long-term goal of using this model for systems biology investigations to better understand human malaria pathogenesis, particularly as it pertains to pathophysiological complications observed in sick patients and with regard to the impact of relapses on both the health of individuals and transmission [
37]. Previous studies have utilized the
P. cynomolgi-macaque model to understand the parasite’s basic biology and parasite kinetics, including the study of hypnozoites and relapses [
20,
33,
38]. However, an extensive characterization of the clinical and haematological perturbations that occur during the infections has not been reported, particularly on a daily basis.
Discussion
This 100-day experiment was designed to study the clinical and parasitological attributes of the primary blood-stage and relapse infections caused by
P. cynomolgi in rhesus macaques and set the stage for future, comprehensive studies using this model to understand the underlying mechanisms of immunity, pathogenesis and disease. Highly resolved kinetics of core haematological parameters were developed after inoculation with infectious B strain sporozoites, and specifically, reticulocyte, platelet, haemoglobin, MPV, and MCV dynamics are presented here. This study expands upon previous rhesus infection studies with the
P. cynomolgi B strain, which primarily monitored parasitological data but did not assess clinical parameters on a daily basis [
17,
32]. The compiled clinical and parasitological datasets generated here provide a glimpse into the daily dynamics of malaria beyond what is typically feasible in humans. It is anticipated that data generated in this experiment (all of which are being shared publicly—see “
Methods” section and Additional file
1) will aid the development and testing of hypotheses by the research community whether for clinical studies or continued investigations using
P. cynomolgi as a model.
Anaemia ranged from mild to severe during the primary infections in this study and appears to be the result of multiple processes. First, it is noteworthy that haemoglobin decreased to the lowest levels after parasitaemia was reduced, whether from the natural control of the infection by the macaque or drug treatment. This data demonstrates that the loss of RBCs due to parasitism is not the sole reason for anaemia in this model. Indeed, other non-human primate malaria models [
43], rodent malaria models [
13] and humans [
50,
51] show a similar kinetic where anaemia is worse after the peripheral parasitaemia has decreased. This phenomenon is attributed to the simultaneous removal of iRBCs and uninfected RBCs [
52,
53], which has also been modelled mathematically to arrive at similar conclusions [
50]. Secondly, the normal, timely replenishment of circulating RBCs by the release of reticulocytes from the bone marrow was disrupted, as shown previously with
P. coatneyi infection of rhesus macaques [
43]. Thus, the disruption of compensatory bone marrow mechanisms with the effective release and/or production of reticulocytes from the bone marrow is likewise apparent in the
P. cynomolgi-rhesus macaque model during the acute, primary infection. For each of these species, which model
P. falciparum and
P. vivax, respectively, the disruption of normal bone marrow physiology could be potentially due to ongoing immunological responses against the parasite or parasite byproducts, such as haemozoin, that are released as the parasites multiply in the blood [
13,
54‐
56]. In agreement as shown here, there was an initial increase of reticulocytes early during the primary infections and these cells returned to baseline levels when the parasitaemia was increasing exponentially in the blood. This supports the view that the disruption of the normal compensatory mechanisms is dependent on parasite levels. Future studies will be needed to better understand the host-pathogen interactions that occur during the primary infection and how they contribute to malarial anaemia in this model.
The relative contribution of thrombocytopaenia to disease outcome during malaria remains controversial and also not well understood [
14,
44,
47]. In this study, all animals developed mild thrombocytopaenia irrespective of their clinical presentations, and thus, thrombocytopaenia was not viewed as an indicator of disease severity or outcome. Prior evidence suggests that thrombocytopaenia could be due to removal of platelets by splenic macrophages during infection [
16], whereas other models suggest that platelets form clumps with iRBCs [
57] or become sequestered in the microvasculature by adhering to activated endothelial cells [
58]. Interestingly, published experiments with the
P. cynomolgi B strain have demonstrated that thrombocytopaenia occurs in both spleen-intact and splenectomized macaques, suggesting that there are other contributing factors in the development of this complication aside from processes associated with this organ [
59]. In agreement, the data from this study indicates that platelet production may have been impaired since the MPV was decreased as the number of platelets rebounded after the peak of parasitaemia. Previous studies with malaria patients have reported changes in MPV during malaria [
16,
60]. The alteration in MPV may indicate disruption of megakaryocyte function, and thus, platelet production. In further support of this hypothesis, both reticulocytes and platelets returned to pre-infection levels together, and since both of these cell types originate from the bone marrow, the simultaneous return to normal levels could be explained by the restoration of normal bone marrow physiology. Overall, these data suggest that the disruption of normal bone marrow physiology during acute malaria may also lead to impaired platelet production and contribute to thrombocytopaenia. Future studies should explore this hypothesis further and aim to understand if thrombocytopaenia is, in part, a bystander effect of bone marrow dysfunction.
It is important to recognize that macaques infected with
P. cynomolgi presented with inter-individual differences in disease severity, similar to humans. In RFv13, a very high parasitaemia (19.5 % parasitaemia), appeared to contribute to the lethality since this parasitaemia clearly distinguished this animal from the two non-severe (RSb14 and RIc14) and two severe (RMe14 and RFa14) clinical presentations. These phenotypes did not clearly stratify away from each other based on parasitological profiles alone, indicating that while parasitaemia may play a role in severe disease, it is not solely responsible for the observed differences in clinical presentation and other pathological processes may come into play. This is reminiscent of human malaria where individuals can progress to severe malaria regardless of parasitaemia [
61‐
63]. Curiously, unlike the four surviving macaques, RFv13 did not show a modest increase in reticulocytes during the beginning of the primary infection. In future studies, it would be useful to evaluate this finding and other potential indicators of the progression of infections and how these early responses to blood-stage infections influence pathogenesis and clinical presentation.
Schmidt [
32] demonstrated that relapse frequencies in the rhesus macaque differ based on the number of
P. cynomolgi B strain sporozoites inoculated, and based on this evidence, some investigators have used 1 × 10
6 sporozoites to generate early, frequent and uniform relapse patterns across individuals for screening anti-hypnozoite drugs [
19]. In contrast, only about 2000 sporozoites were inoculated in the current study, aiming for primary infections followed by more natural patterns of relapse. While this is still higher than what is suspected to be naturally injected by a mosquito [
64,
65], 2000 sporozoites best ensured at least one or two relapses in all animals during the course of this study. In fact, the average number of relapses observed was 1.5. This number may have been higher if the animals had not been cured as a group after the fifth specimen collection. Nevertheless, the observed relapse patterns reported here are similar to what would be expected with
P. vivax infections with tropical strains of this parasite [
7,
66,
67]. A follow-up, iterative experiment was designed to monitor the natural relapse patterns, without such treatment plans, and to monitor gametocytes during the relapse periods more carefully.
Understanding, preventing and treating relapses caused by
P. vivax, and also
P. ovale, remain key challenges in today’s malaria eradication efforts, especially if asymptomatic carriers remain infectious to mosquitoes. Critically, the relative impact of primary
versus relapse clinical malaria presentations has remained undefined in a controlled, experimental model system and direct evidence from human studies is not available because of the challenges distinguishing the two in human cases without a well-controlled study design [
68‐
71]. Here, each
bona fide relapse, subsequent to blood-stage treatment and in the absence of re-infections, resulted in significantly lower parasitaemias compared to the primary infections and minimal, if any, changes in clinical parameters to indicate illness. This suggests that immunity, or potentially other undefined mechanisms, during the primary infection, led to controlled relapses characterized by reduced parasitaemias and lack of clinical complications. Whenever minor alterations in clinical parameters were observed during a relapse (e.g., minor drop in haemoglobin levels from 14 to 11 g/dl), as was the case for RMe14, these alterations resolved in a controlled, non-pathological manner, without the need for clinical support. The lack of clinical illness during relapses was not necessarily expected, especially in animals that only experienced a single primary blood-stage infection, since previous reports have concluded, through mathematical and statistical modelling of data collected from holo-endemic areas that relapses are responsible for up to 96 % of blood-stage
P. vivax infections [
11,
72]. Thus, this experiment draws light to the question “What percentage of relapses actually cause clinical malaria?” Relapses have been a major concern due to their role in causing possible repeated bouts of illness and because they can be a source of infectious gametocytes to maintain transmission. The data presented here support the hypothesis that relapses may not necessarily be the main cause of clinical vivax or ovale malaria cases. This warrants further study to understand the clinical outcome of primary infections compared to relapses, in addition to transmissibility questions, caused by homologous or heterologous strains as would also be anticipated in malaria-endemic regions [
71,
73,
74].
In a previous study, rhesus macaques infected with
P. cynomolgi were treated soon after infected RBCs were detected, and the parasitaemia of the first relapses appeared to be higher than subsequent relapses [
19]. The clinical status of the macaques during relapses was not presented, however, so it is unknown if the clinical profile of the relapses differed from the primary infections. In contrast, the parasitaemia in the current study was allowed to persist during the primary infections, since a goal was to understand the course of the clinical presentations. Interestingly, with this approach, the first and second relapses had similar parasitological and clinical attributes, and the difference in parasitaemia between the primary infection and relapses was substantial. These data highlight the point that the timing of the experimental treatment of blood-stage infections may affect the later presentation of relapses, and could in turn influence whether the relapse parasitaemia and illness are suppressed, or not. Future studies should evaluate if the timing of blood-stage treatment affects the development of immunity against the parasite.
As shown by this research team and others using this model system, relapse infections can be definitively produced under controlled experimental conditions [
19,
32], in the absence of new sporozoite-initiated infections, and they can be distinguished from blood-stage recrudescences through the administration of curative blood-stage treatments. This creates a clear ‘window’ of time after the administration of curative treatment of blood-stage infections, during which time the activation of hypnozoites and appearance of relapse parasites can be anticipated and studied. While a challenging prospect, it is during this period that the identification of blood-based, metabolic biomarkers of liver-stage forms may 1 day become a reality, paving the way towards diagnostic tools and curative liver-stage treatments [
75].
Authors’ contributions
Conceived and designed the experiments: MRG, AM, EVSM, and JWB and members of the MaHPIC Consortium. Performed the experiments: CJ and MCM, and members of the MaHPIC consortium. Managed and deposited the data and metadata: JCK and members of the MaHPIC consortium. Performed data analysis and generated the Figures: CJ. Interpreted the data analysis: CJ, AM, JWB, and MRG. Wrote the paper: CJ and MRG. Provided expert knowledge, viewpoints and manuscript contributions: AM, JWB and JCK, and members of the MaHPIC consortium. All authors read and approved the final manuscript.