Most Plasmodium falciparum infections that are detected in community surveys are characterized by low-density parasitaemia and the absence of clinical symptoms. Molecular diagnostics have shown that this asymptomatic parasitic reservoir is more widespread than previously thought, even in low-endemic areas. In this Opinion article, we describe the detectability of asymptomatic malaria infections and the relevance of submicroscopic infections for parasite transmission to mosquitoes and for community interventions that aim at reducing transmission. We argue that wider deployment of molecular diagnostic tools is needed to provide adequate insight into the epidemiology of malaria and infection dynamics to aid elimination efforts.

Clonally variant expression of surface antigens allows the malaria parasite Plasmodium falciparum to evade immune recognition during blood stage infection and secure malaria transmission. We demonstrate that heterochromatin protein 1 (HP1), an evolutionary conserved regulator of heritable gene silencing, controls expression of numerous P. falciparum virulence genes as well as differentiation into the sexual forms that transmit to mosquitoes. Conditional depletion of P. falciparum HP1 (PfHP1) prevents mitotic proliferation of blood stage parasites and disrupts mutually exclusive expression and antigenic variation of the major virulence factor PfEMP1. Additionally, PfHP1-dependent regulation of PfAP2-G, a transcription factor required for gametocyte conversion, controls the switch from asexual proliferation to sexual differentiation, providing insight into the epigenetic mechanisms underlying gametocyte commitment. These findings show that PfHP1 is centrally involved in clonally variant gene expression and sexual differentiation in P. falciparum and have major implications for developing antidisease and transmission-blocking interventions against malaria.

The malaria vaccine Combination B comprises recombinant Plasmodium falciparum ring-infected erythrocyte surface antigen and 2 merozoite surface proteins (MSP1 and MSP2) formulated in oil-based adjuvant. A phase 1-2b double-blind, randomized, placebo-controlled trial in 120 children (5-9 years old) in Papua New Guinea demonstrated a 62% (95% confidence limits: 13%, 84%) reduction in parasite density in children not pretreated with sulfadoxine-pyrimethamine. Vaccinees had a lower prevalence of parasites carrying the MSP2-3D7 allelic form (corresponding to that in the vaccine) and a higher incidence of morbid episodes associated with FC27-type parasites. These results demonstrate functional activity of Combination B against P. falciparum in individuals with previous malaria exposure. The specific effects on parasites with particular msp2 genotypes suggest that the MSP2 component, at least in part, accounted for the activity. The vaccine-induced selection pressure exerted on the parasites and its consequences for morbidity strongly argue for developing vaccines comprising conserved antigens and/or multiple components covering all important allelic types.

BackgroundPlanning and evaluating malaria control strategies relies on accurate definition of parasite prevalence in the population. A large proportion of asymptomatic parasite infections can only be identified by surveillance with molecular methods, yet these infections also contribute to onward transmission to mosquitoes. The sensitivity of molecular detection by PCR is limited by the abundance of the target sequence in a DNA sample; thus, detection becomes imperfect at low densities. We aimed to increase PCR diagnostic sensitivity by targeting multi-copy genomic sequences for reliable detection of low-density infections, and investigated the impact of these PCR assays on community prevalence data.Methods and FindingsTwo quantitative PCR (qPCR) assays were developed for ultra-sensitive detection of Plasmodium falciparum, targeting the high-copy telomere-associated repetitive element 2 (TARE-2, ∼250 copies/genome) and the var gene acidic terminal sequence (varATS, 59 copies/genome). Our assays reached a limit of detection of 0.03 to 0.15 parasites/μl blood and were 10× more sensitive than standard 18S rRNA qPCR. In a population cross-sectional study in Tanzania, 295/498 samples tested positive using ultra-sensitive assays. Light microscopy missed 169 infections (57%). 18S rRNA qPCR failed to identify 48 infections (16%), of which 40% carried gametocytes detected by pfs25 quantitative reverse-transcription PCR. To judge the suitability of the TARE-2 and varATS assays for high-throughput screens, their performance was tested on sample pools. Both ultra-sensitive assays correctly detected all pools containing one low-density P. falciparum–positive sample, which went undetected by 18S rRNA qPCR, among nine negatives. TARE-2 and varATS qPCRs improve estimates of prevalence rates, yet other infections might still remain undetected when absent in the limited blood volume sampled.ConclusionsMeasured malaria prevalence in communities is largely determined by the sensitivity of the diagnostic tool used. Even when applying standard molecular diagnostics, prevalence in our study population was underestimated by 8% compared to the new assays. Our findings highlight the need for highly sensitive tools such as TARE-2 and varATS qPCR in community surveillance and for monitoring interventions to better describe malaria epidemiology and inform malaria elimination efforts.

Carriage and density of gametocytes, the transmission stages of malaria parasites, are determined for predicting the infectiousness of humans to mosquitoes. This measure is used for evaluating interventions that aim at reducing malaria transmission. Gametocytes need to be detected by amplification of stage-specific transcripts, which requires RNA-preserving blood sampling. For simultaneous, highly sensitive quantification of both, blood stages and gametocytes, we have compared and optimized different strategies for field and laboratory procedures in a cross sectional survey in 315 5-9 yr old children from Papua New Guinea. qRT-PCR was performed for gametocyte markers pfs25 and pvs25, Plasmodium species prevalence was determined by targeting both, 18S rRNA genes and transcripts. RNA-based parasite detection resulted in a P. falciparum positivity of 24.1%; of these 40.8% carried gametocytes. P. vivax positivity was 38.4%, with 38.0% of these carrying gametocytes. Sensitivity of DNA-based parasite detection was substantially lower with 14.1% for P. falciparum and 19.6% for P. vivax. Using the lower DNA-based prevalence of asexual stages as a denominator increased the percentage of gametocyte-positive infections to 59.1% for P. falciparum and 52.4% for P. vivax. For studies requiring highly sensitive and simultaneous quantification of sexual and asexual parasite stages, 18S rRNA transcript-based detection saves efforts and costs. RNA-based positivity is considerably higher than other methods. On the other hand, DNA-based parasite quantification is robust and permits comparison with other globally generated molecular prevalence data. Molecular monitoring of low density asexual and sexual parasitaemia will support the evaluation of effects of up-scaled antimalarial intervention programs and can also inform about small scale spatial variability in transmission intensity.

BackgroundThe undetectable hypnozoite reservoir for relapsing Plasmodium vivax and P. ovale malarias presents a major challenge for malaria control and elimination in endemic countries. This study aims to directly determine the contribution of relapses to the burden of P. vivax and P. ovale infection, illness, and transmission in Papua New Guinean children.Methods and FindingsFrom 17 August 2009 to 20 May 2010, 524 children aged 5–10 y from East Sepik Province in Papua New Guinea (PNG) participated in a randomised double-blind placebo-controlled trial of blood- plus liver-stage drugs (chloroquine [CQ], 3 d; artemether-lumefantrine [AL], 3 d; and primaquine [PQ], 20 d, 10 mg/kg total dose) (261 children) or blood-stage drugs only (CQ, 3 d; AL, 3 d; and placebo [PL], 20 d) (263 children). Participants, study staff, and investigators were blinded to the treatment allocation. Twenty children were excluded during the treatment phase (PQ arm: 14, PL arm: 6), and 504 were followed actively for 9 mo. During the follow-up time, 18 children (PQ arm: 7, PL arm: 11) were lost to follow-up. Main primary and secondary outcome measures were time to first P. vivax infection (by qPCR), time to first clinical episode, force of infection, gametocyte positivity, and time to first P. ovale infection (by PCR). A basic stochastic transmission model was developed to estimate the potential effect of mass drug administration (MDA) for the prevention of recurrent P. vivax infections. Targeting hypnozoites through PQ treatment reduced the risk of having at least one qPCR-detectable P. vivax or P. ovale infection during 8 mo of follow-up (P. vivax: PQ arm 0.63/y versus PL arm 2.62/y, HR = 0.18 [95% CI 0.14, 0.25], p < 0.001; P. ovale: 0.06 versus 0.14, HR = 0.31 [95% CI 0.13, 0.77], p = 0.011) and the risk of having at least one clinical P. vivax episode (HR = 0.25 [95% CI 0.11, 0.61], p = 0.002). PQ also reduced the molecular force of P. vivax blood-stage infection in the first 3 mo of follow-up (PQ arm 1.90/y versus PL arm 7.75/y, incidence rate ratio [IRR] = 0.21 [95% CI 0.15, 0.28], p < 0.001). Children who received PQ were less likely to carry P. vivax gametocytes (IRR = 0.27 [95% CI 0.19, 0.38], p < 0.001). PQ had a comparable effect irrespective of the presence of P. vivax blood-stage infection at the time of treatment (p = 0.14). Modelling revealed that mass screening and treatment with highly sensitive quantitative real-time PCR, or MDA with blood-stage treatment alone, would have only a transient effect on P. vivax transmission levels, while MDA that includes liver-stage treatment is predicted to be a highly effective strategy for P. vivax elimination. The inclusion of a directly observed 20-d treatment regime maximises the efficiency of hypnozoite clearance but limits the generalisability of results to real-world MDA programmes.ConclusionsThese results suggest that relapses cause approximately four of every five P. vivax infections and at least three of every five P. ovale infections in PNG children and are important in sustaining...

BackgroundWhere P. vivax and P. falciparum occur in the same population, the peak burden of P. vivax infection and illness is often concentrated in younger age groups. Experiences from malaria therapy patients indicate that immunity is acquired faster to P. vivax than to P. falciparum challenge. There is however little prospective data on the comparative risk of infection and disease from both species in young children living in co-endemic areas.Methodology/Principal FindingsA cohort of 264 Papua New Guinean children aged 1-3 years (at enrolment) were actively followed-up for Plasmodium infection and febrile illness for 16 months. Infection status was determined by light microscopy and PCR every 8 weeks and at each febrile episode. A generalised estimating equation (GEE) approach was used to analyse both prevalence of infection and incidence of clinical episodes. A more pronounced rise in prevalence of P. falciparum compared to P. vivax infection was evident with increasing age. Although the overall incidence of clinical episodes was comparable (P. falciparum: 2.56, P. vivax 2.46 episodes / child / yr), P. falciparum and P. vivax infectious episodes showed strong but opposing age trends: P. falciparum incidence increased until the age of 30 months with little change thereafter, but incidence of P. vivax decreased significantly with age throughout the entire age range. For P. falciparum, both prevalence and incidence of P. falciparum showed marked seasonality, whereas only P. vivax incidence but not prevalence decreased in the dry season.Conclusions/SignificanceUnder high, perennial exposure, children in PNG begin acquiring significant clinical immunity, characterized by an increasing ability to control parasite densities below the pyrogenic threshold to P. vivax, but not to P. falciparum, in the 2nd and 3rd year of life. The ability to relapse from long-lasting liver-stages restricts the seasonal variation in prevalence of P. vivax infections.

Malaria infections occurring below the limit of detection of standard diagnostics are common in all endemic settings. However, key questions remain surrounding their contribution to sustaining transmission and whether they need to be detected and targeted to achieve malaria elimination. In this study we analyse a range of malaria datasets to quantify the density, detectability, course of infection and infectiousness of subpatent infections. Asymptomatically infected individuals have lower parasite densities on average in low transmission settings compared to individuals in higher transmission settings. In cohort studies, subpatent infections are found to be predictive of future periods of patent infection and in membrane feeding studies, individuals infected with subpatent asexual parasite densities are found to be approximately a third as infectious to mosquitoes as individuals with patent (asexual parasite) infection. These results indicate that subpatent infections contribute to the infectious reservoir, may be long lasting, and require more sensitive diagnostics to detect them in lower transmission settings.