Background
Plasmodium parasites are the causative agents of human malaria. Infection begins when female
Anopheles mosquitoes take a blood meal. During feeding, mosquitoes transmit sporozoite-stage parasites into the dermis. Sporozoites make their way to blood vessels and then to the liver, where they develop over the next several days. The sporozoite and liver stage parasites are clinically silent. At the completion of the liver stage, parasites are released into the bloodstream and invade erythrocytes. The resulting cyclical infection of erythrocytes is responsible for all clinical disease. During the erythrocyte stage, parasites can be detected in whole blood using several diagnostic tests such as microscopy of Giemsa-stained blood smears, lateral flow rapid diagnostic tests for parasite antigens and nucleic acid tests (NATs). In general, NATs are more analytically sensitive than other modalities [
1]. The most common NAT targets are DNA genes encoding the
Plasmodium 18S ribosomal RNAs (hereafter called 18S rDNA) or the 18S rRNAs themselves, with testing by polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR), respectively [
1].
Plasmodium 18S rRNA/rDNA-targeted NATs are intended to detect blood-stage parasites, but they also can detect the same sequences in sporozoite and liver stages. Most evidence suggests that sporozoites transit from the mosquito inoculation site to the liver in less than an hour [
2]. However, if sporozoite-derived nucleic acids, or even sporozoites, continued to circulate for days following sporozoite exposure, these nucleic acids could generate false positive results for NATs intended to monitor for erythrocyte infection. An earlier study reported this type of NAT positivity in mice [
3]. Abkallo and colleagues reported that
Plasmodium yoelii 18S rDNA was detectable in peripheral mouse blood by qPCR after
P. yoelii sporozoite injection, but before emergence of infected erythrocytes from the liver at about 48 h. Compared to
Plasmodium falciparum,
P. yoelii has a shorter liver stage with erythrocyte stage emergence ~ 48 h post-inoculation. In the Abkallo study, CBA mice were infected with 2.5 × 10
4 P. yoelii sporozoites by tail vein injection, and blood was sampled by capillary action from the tail vein at later time points. For 90 min following
P. yoelii sporozoite injection, 18S rDNA was detected in tail vein peripheral blood at low and decreasing concentrations. 18S rRNA was next detected ~ 24 h post-injection (at ~ 50–100 copies of 18S rDNA/μL of blood) followed by a decrease to baseline and then a marked rise at 48 h corresponding with erythrocyte stage infection and blood smear positivity. Based on these data, the authors concluded that NAT positivity prior to the emergence of erythrocyte stage parasites was from circulating pre-erythrocytic parasites. Such a result could complicate the use of
Plasmodium 18S rRNA/rDNA NATs to assess infection in pre-clinical and clinical trials when using attenuated sporozoite vaccines that must be monitored for safety or using wild-type sporozoites for challenge studies [
4]. Thus, to further investigate whether pre-erythrocytic parasites are a confounder of peripheral blood NATs, additional experiments using the 18S rRNA biomarker were conducted in mice and non-human primates (NHP).
Discussion
The
Plasmodium 18S rRNA/rDNA biomarker is a sensitive diagnostic marker that is able to achieve earlier detection of infection compared to blood smears [
7,
12]. However, conflicting evidence about the persistence of sporozoite-derived 18S rRNA led to concerns about the potential for false positive results due to persistently circulating sporozoites or their byproducts.
Here, studies in mice demonstrated that P. yoelii sporozoite 18S rRNA does not persistently circulate in peripheral blood and suggests that timing and concentration of the pre-erythrocytic 18S rDNA positivity 24 h post-challenge in the Abkallo study may have been due to re-sampling of locally deposited contaminating 18S rDNA. Direct data comparison between studies is limited since the assay used in the Abkallo study was a DNA-only test for Plasmodium 18S rRNA genes. However, in agreement with that report, in this study low amounts of Plasmodium 18S rRNA/rDNA could be detected. The rRNA versus rDNA comparison performed here suggests that this signal was likely from residual, locally deposited contaminating P. yoelii 18S rDNA. Thus, the 18S rRNA approach to parasite detection was advantageous since such tiny amounts of nucleic acid template do not constitute the 18S rRNA content of a single parasite and would have been deemed negative by 18S rRNA assays even if blood was collected from the tail vein site. In 50 μL blood samples obtained distally from the inoculation site in mice, there was no evidence of peripheral circulation of P. yoelii sporozoite-derived 18S rRNA 1 day after administration. It is also notable that these cardiac puncture blood samples contained 10–20 times more volume of blood per sample than the tail vein-collected dried blood spots, further supporting the conclusion that there is no long lasting, peripherally circulating P. yoelii 18S rRNA during the liver stage.
Studies in rhesus macaques also showed no evidence for persistent
P. falciparum 18S rRNA circulation following administration of an exceptionally high dose of freshly-dissected PfSPZ. The PfSPZ dose given to NHP (6.5 × 10
6) was > 2000-fold higher than the standard 3.2 × 10
3 PfSPZ of Sanaria
® PfSPZ Challenge (aseptic, purified, cryopreserved PfSPZ) that always cause blood stage parasitemia in human subjects [
13,
14], and comparably higher than the standard five mosquito bite dose used for controlled human malaria infections (CHMI) [
15]. The NHP data support the conclusion that
P. falciparum parasites and their 18S rRNAs do not circulate in the days following
P. falciparum sporozoite inoculation. These data, coupled with the relatively small number of sporozoites delivered by infected mosquito bites or by PfSPZ Challenge and the intended timing of diagnostic sample collection starting on day 6 or later, make the risk of sporozoite-induced false positives negligible in human challenge studies.
In addition, the NHP studies are consistent with expansion of liver-stage parasites (roughly 101-fold from day 3 to 6 post-infection) in these animals. While immunogenicity of PfSPZ vaccines has been assessed in rhesus [
16], pre-erythrocytic protection studies are usually not tested in rhesus because they do not develop blood-stage
P. falciparum infections after receiving
P. falciparum sporozoites [
11]. However,
P. falciparum sporozoites invade many different cell types [
17] including rhesus hepatocytes in vitro (albeit with lower efficiency than human hepatocytes) [
9,
10].
Plasmodium falciparum sporozoites that successfully invade rhesus hepatocytes in vitro subsequently express PfEXP1, a protein not expressed in sporozoites [
9], indicating that the parasites continue to develop in these cells. Wild-type
P. falciparum sporozoites in human hepatocytes proliferate ~ 30,000-fold during their 6.5 day development [
2] with most proliferation occurring in the latter part of the cycle. The 101-fold increase observed from day 3 to 6 in rhesus macaques in this study demonstrates proliferation of
P. falciparum in infected rhesus hepatocytes, though the current data does not match the proliferative potential of
P. falciparum as measured in human liver. This study was limited by the small number of NHP (n = 2 animals per timepoint) and a relatively small number of liver tissue samples overall. More systematic liver sampling using larger biopsies collected throughout the pre-erythrocytic stage could be useful for understanding and measuring the full growth potential of the
P. falciparum liver stage in rhesus macaques. The data also suggest that rhesus could be further investigated for use in testing pre-erythrocytic stage-targeted vaccines and/or drugs using a liver-stage
P. falciparum 18S rRNA endpoint as a measure of efficacy.
In agreement with the mouse and NHP findings presented herein, CHMI studies also support the lack of circulating
P. falciparum sporozoite-derived 18S rRNA after
P. falciparum sporozoite inoculation.
Plasmodium falciparum 18S rRNA was not detected 7, 10 or 28 days after administration of genetically-attenuated GAP3KO sporozoites delivered by 150–200
P. falciparum GAP3KO-infected mosquito bites [
18]. The GAP3KO study is highly relevant to the question of persistent circulating 18S rRNA/rDNA since the attenuated parasite does not lead to formation of infected erythrocytes so any circulating
P. falciparum 18S rRNA would have been presumed to be
P. falciparum sporozoite-derived.
Like the animal studies presented herein, rising biomarker positivity from day 6 to 7 post-infection in CHMI studies at multiple centres [
7,
19‐
23] strongly supports the well-studied timing of erythrocyte stage emergence from the human liver and suggests that testing starting on days 6–7 post-inoculation is appropriate in human clinical trials. These findings are further corroborated by earlier studies aimed at measuring the duration of the liver stage by culturing parasites during days 5–9.5 post-inoculation [
24] where
P. falciparum could only be cultured from
P. falciparum sporozoite-infected human volunteers from day 6.5 onward. In another early study, human volunteers were bitten by infected mosquitoes and their blood was then sub-inoculated into different human recipients at timepoints thereafter [
2].
Plasmodium falciparum infections could only be successfully sub-inoculated into recipients when donor blood was collected within 1 h of the donor’s original mosquito bites and not again until 5–6 days later when the erythrocyte stage had begun. The overall conclusion is that in the days that follow sporozoite exposure, sporozoites do not circulate in peripheral blood. Therefore, the data collectively indicate that in humans the presence of
P. falciparum 18S rRNA days after sporozoite inoculation reflects
P. falciparum erythrocyte stage parasite emergence, not the persistence of
P. falciparum sporozoite-derived 18S rRNA.
Authors’ contributions
SCM, TMO, ASI, ZPB, AMS, NS, and EC performed the experiments. SCM, SC, BKLS, SK, SLH and RAS provided reagents. SCM and MC wrote the manuscript. All authors reviewed the data. All authors read and approved the final manuscript.