Background
A significant proportion of individuals living in areas where malaria is endemic have
Plasmodium parasitaemia without the presence of symptoms. This is thought to reflect either the early stages of infection or the acquisition of clinical immunity that restricts parasite expansion and prevents clinical disease [
1,
2]. Microscopy or rapid diagnostic tests remain the most common method for the diagnosis of
Plasmodium infection. However, it is now recognized that even in low-transmission settings a large proportion of microscopy-negative individuals have sub-microscopic parasitaemia identified by polymerase chain reaction (PCR) [
3‐
7]. These sub-microscopic infections frequently remain undetected and untreated and hence, contribute significantly to the transmittable reservoir and potentially to morbidity [
8]. Sensitive molecular techniques are now used increasingly in epidemiological studies to detect sub-microscopic infection with limits of detection below 0.1 parasites per μl [
9‐
12]. Sub-microscopic
Plasmodium infections are important and warrant consideration in malaria intervention and elimination programmes [
9,
13].
While immune correlates in microscopy-positive asymptomatic parasitaemia have been reported [
14‐
17], few data exist on immune regulation at very low parasitaemia, with no data available on the dendritic cell (DC) and regulatory T (Treg) cell response at sub-microscopic levels of infection in malaria-endemic areas. In individuals hospitalized with clinical disease from
Plasmodium falciparum and
Plasmodium vivax, blood DC are functionally and numerically compromised [
18,
19] while Treg cells expand [
20,
21], promoting immune suppression. Similarly, in low-dose experimental
P. falciparum blood-stage infection of naïve volunteers, blood DC become non-functional and apoptose at PCR-level parasitaemia below the detection limit of microscopy [
22,
23]. In contrast, the recent demonstration that adults and children with asymptomatic microscopy-positive
P. falciparum or
P. vivax infection have increased or retained activation of blood DC and reduced activation of Treg cells [
14], suggests that appropriate DC maturation and less Treg suppression may contribute to the active control of asymptomatic infection. Therefore, in contrast to naïve individuals with sub-microscopic infection, it is hypothesized that asymptomatic-exposed individuals with sub-microscopic infection respond similarly to patent asymptomatic-exposed individuals, such that DC remain functional while Treg cells display reduced suppressive function.
In a previous publication [
14], parasitaemia in asymptomatic adults was evaluated by microscopy only. Using PCR, a large proportion of microscopy-negative controls were subsequently identified as having sub-microscopic
Plasmodium infection. In an extension of the original study [
14], flow cytometry data were re-categorized in adults using the additional PCR results. Sub-microscopic infection with
P. falciparum or
P. vivax was identified from microscopy-negative controls and then compared to patent infection and acute malaria. The results indicate that while peripheral CD4
+ T cells decline, blood DC and Treg cells remain numerically and phenotypically, functionally preserved during asymptomatic sub-microscopic
P. falciparum or
P. vivax infection.
Discussion
As in previous studies in Southeast Asia [
4,
5] a large proportion of adults living in Timika have sub-microscopic infection with
P. falciparum or
P. vivax. In these individuals HLA-DR expression on total DC is retained. Similarly, the Treg cell phenotype, characterized by aTreg frequencies and rTreg-to-aTreg ratios, was comparable to that in uninfected adult controls. Collectively, the data indicate that neither DC nor Treg cells are phenotypically dysregulated by asymptomatic sub-microscopic
Plasmodium infection. The absence of dysregulation suggests functional potential of these cells is maintained and may contribute to the control of infection and clinical immunity to
Plasmodium infection.
The expression of HLA-DR is required by specialist antigen-presenting cells, such as DC, to present foreign antigen to T cells and is commonly used as a marker for DC activation/maturation. In controlled human
Plasmodium infection studies, naïve volunteers experimentally infected with
P. falciparum displayed reduced circulating pDC and mDC levels, followed by down-regulation of HLA-DR on the surface of pDC [
22] and CD1c
+ mDC [
23], all occurring at sub-microscopic PCR-level parasitaemia prior to development of patent parasitaemia. Conversely, in both sub-microscopic and patent asymptomatic infections in endemic area residents, the observation of unaltered HLA-DR expression on total circulating DC indicates that DC maturation is not reduced, which may contribute to the maintenance of asymptomatic infection. Comprehensive longitudinal studies in asymptomatic adults are required to fully characterize DC activation/function and to understand their stability and role in the maintenance of sub-microscopic and patent infection.
In contrast to DC activation, Treg cell activation may contribute to progression of disease by suppressing T cell responses and host immunity, favouring parasite growth [
20,
21,
28]. CD25
hiCD45RA
−-activated Treg cells (aTreg) are highly suppressive and proliferative Treg cells [
29], while CD25
+CD45RA
+-resting Treg cells (rTreg) provide a reservoir capable of subsequent activation. Adults with asymptomatic sub-microscopic
P. falciparum or
P. vivax infection showed neither a reduced nor an over-activated Treg cell response, characterized by aTreg frequencies and rTreg-to-aTreg ratios that were comparable to uninfected adult controls. This contrasted with the reduced Treg cell response characterized by a relative decrease in aTreg frequency and increase in rTreg-to-aTreg ratio in asymptomatic patent
Plasmodium infection [
14]. Furthermore, the data contrast with the more activated Treg cells observed in acute malaria [
14,
20]. Therefore, in asymptomatic sub-microscopic infections, the data indicate that the CD45RA phenotype of Treg cells was not modulated and suggests the Treg cell compartment is phenotypically altered only by patent and not sub-microscopic parasitaemia. This suggests maintenance of a normal, stably suppressive environment, allowing other immune cells to contribute to the control of infection.
Correlations between parasitaemia and Treg cells could not be examined since the degree of sub-microscopic parasitaemia was not quantified. Boyle and colleagues [
17] recently showed the loss of circulating Treg cells and down-regulation of TNFRII in Ugandan children highly exposed to malaria, which is thought to be implicated in the development of protective immunity to malaria. Additional studies are warranted to examine relationships between Treg cell activation and parasitaemia, including additional Treg cell activation markers (such as TNFRII, CTLA-4), and longitudinal assessment of Treg cell responses in asymptomatic carriers.
In sub-microscopic
P. falciparum infection, peripheral lymphocytes and CD4
+ T cells were reduced in asymptomatic adults, a finding similar to that observed in experimental sub-microscopic
P. falciparum infection of naïve volunteers [
22]. Despite this, no change in Treg cell frequency and numbers were found in endemic area residents with asymptomatic sub-microscopic
P. falciparum or
P. vivax infection, suggesting that stable Treg cells potentially minimize immune suppression and thereby support immune activation. Although asymptomatic adults with sub-microscopic
P. falciparum infection were lymphopenic, their lymphocyte and CD4
+ T cell counts were still significantly higher compared to adults with species-matched acute malaria.
The study had a number of limitations. As patent individuals were treated and sub-microscopic infections were only retrospectively identified, participants could not be longitudinally followed in this study, preventing the assessment of whether infected individuals remained asymptomatic, cleared the infection, or developed symptomatic malaria. It is also acknowledged that the sample sizes in the asymptomatic sub-microscopic groups with DC data were low due to poor sample quality and/or limitations in available blood volume. In addition, PCR was not performed on the sub-set of children in the original study [
14] because finger-prick blood volumes were used for whole blood flow cytometry and this left insufficient or no blood for molecular analysis. Despite these limitations, modulations in DC and Treg cells clearly exist between controls, patent infection and acute malaria [
14]. The current data indicate that sub-microscopic parasitaemia in endemic adults is not associated with variations in DC HLA-DR or Treg cell CD45RA expression.
Authors’ contributions
SK, TW, GM, KAP and NMA conceived and designed the study; SK, IH, KAP, RN, AK, FHB, EK, DAL and JRP carried out household survey and patient recruitment; SK performed field laboratory experiments; IH, ZP, RN, AK, JM and RNP performed and analysed PCR experiments: SK, TW and GM analysed flow cytometry data and statistics; SK, TW, GM, JM, RN, NMA, CRE, JRP and RNP: intellectual contribution and preparation of manuscript. All authors read and approved the final manuscript.